Ehrlichial invasin for immunization, diagnosis, and cell delivery

ABSTRACT

Disclosed are vaccines containing one or more immunogenic polypeptides derived from an EtpE protein from an  Ehrlichia  sp. or nucleic acid encoding these polypeptides. Also disclosed is a method for vaccinating a subject against  Ehrlichia  sp. that involves administering to the subject a composition comprising any of the disclosed vaccines. Also disclosed is a method for diagnosing and/or monitoring the treatment of Ehrlichiosis in a subject that comprising assaying a biological sample (e.g., blood, serum, or plasma sample) from the subject for the presence of an antibody that specifically binds an EtpE polypeptide. Also disclosed are methods for delivering a therapeutic or diagnostic agent to a cell in a subject that involves conjugating the agent, or a delivery vehicle comprising the agent, to polypeptide containing the C-terminal domain of an EtpE protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.61/732,491, filed Dec. 3, 2012, and Application Ser. No. 61/810,039,filed Apr. 9, 2013, which are hereby incorporated herein by reference intheir entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government Support under Grant No.R01AI30010 and Grant No. R01AI047885 awarded by the National Institutesof Health. The Government has certain rights in the invention.

BACKGROUND

Ehrlichia chaffeensis causes human monocytic ehrlichiosis (HME), anemerging tick-borne zoonosis. From the site of infected tick bite onhuman skin, E. chaffeensis infects monocytes and spreads via thebloodstream to various tissues, causing a systemic febrile disease. HMEis characterized by fever, headache, myalgia, thrombocytopenia,leucopenia, and elevated liver-enzyme levels, but complications such aspulmonary insufficiency, renal failure, encephalopathy, and disseminatedintravascular coagulation can cause death [Paddock C D, et al. (2003)Clin Microbiol Rev 16: 37-64]. Early diagnosis and the proper treatmentwith doxycycline are critical to prevent complications. The disease isof particular threat to the immunocompromised and the elderly people[Paddock C D, et al. (2003) Clin Microbiol Rev 16: 37-64].

E. chaffeensis is a small obligatory intracellular bacterium. It belongsto the family Anaplasmataceae in the order Rickettsiales that includesmany understudied pathogens of veterinary and public health importance[Rikihisa Y (2010) Nat Rev Microbiol 8: 328-339]. By electronmicroscopy, E. chaffeensis is a polymorphic bacterium (0.2-2.0 μm indiameter), and can be morphologically categorized as small dense-coredcells (DCs) or large reticulate cells (RCs) [Popov V L, et al. (1995) JMed Microbiol 43: 411-421]. DCs are approximately 0.2-0.5 μm indiameter, which is close to the size of the elementary body of Chlamydiaand larger viruses such as Vaccinia virus. By light microscopy, it isnot possible to distinguish individual RCs and DCs, since E. chaffeensisaggregates inside eukaryotic host cells. The characteristic clump ofintracellular E. chaffeensis organisms is termed as “morula” (mulberryin Latin) [Rikihisa Y (2010) Nat Rev Microbiol 8: 328-339]. However,when they are freshly isolated from host cells and dispersed, smallerbacteria (<0.5 μm) are more densely stained with basic dye than largerbacteria (>0.5 μm); therefore, they were defined as DCs and RCs,respectively [Zhang J Z, et al. (2007) Cell Microbiol 9: 610-618]. DCsare more resistant to strong sonication and more infectious than RCs[Cheng Z, et al. (2008) J Bacteriol 190: 2096-2105]. In cell culture, abiphasic developmental cycle has been reported: initially smallinfectious DCs bind to and internalize into host cells, which thendevelop into larger replicating RCs inside a membrane-lined compartmentthat resembles early endosomes. After replication in expandinginclusions, the mature RCs transform back into DCs prior to release fromthe host cells [Zhang J Z, et al. (2007) Cell Microbiol 9: 610-618;Cheng Z, et al. (2008) J Bacteriol 190: 2096-2105]. In patients' bloodspecimens, monocytes were primarily infected with E. chaffeensis, andhence, the disease was named as “monocytic ehrlichiosis” to distinguishit from “granulocytic ehrlichiosis” caused by infection withgranulocyte-tropic Ehrlichia sp. [Paddock C D, et al. (2003) ClinMicrobiol Rev 16: 37-64]. E. chaffeensis can replicate well in severalmammalian cell lines including canine histiocytic leukemia (DH82), humanacute leukemia (THP-1), human promyelocytic leukemia (HL-60), humanembryonic kidney (HEK293), and monkey endothelial (RF/6A) cells [Mott J,et al. (1999) Infect Immun 67: 1368-1378; Liu H, et al. (2012) CellMicrobiol 14: 1037-1050; Miura K, et al. (2011) Infect Immun 79:4947-4956].

Entry into the permissive eukaryotic host cells is essential for E.chaffeensis to sustain its life, since its small genome of 1.18 Mb lacksa large portion of metabolic genes that are required for free living[Dunning Hotopp J C, et al. (2006) PLoS Genet 2:e21]. E. chaffeensisalso lacks LPS, peptidoglycan, lipoteichoic acid, and flagella thatengage Toll-like or NOD-like receptors, or scavenger receptors [RikihisaY (2010) Nat Rev Microbiol 8: 328-339; Rikihisa Y (2010) Nat RevMicrobiol 8: 328-339]. E. chaffeensis entry and subsequent infection ofTHP-1 cells, but not binding are almost completely inhibited bymonodansylcadaverine (MDC), a transglutaminase inhibitor [Lin M, et al.(2002) Infect Immun 70: 889-898]. MDC is known to block Neorickettsiaristicii (formerly Ehrlichia risticii) entry and infection of P388D1cells, vesicular stomatitis virus uptake and receptor-mediatedendocytosis of α2-macroglobulin by Swiss 3T3 mouse cells, but not theuptake of latex beads by P388D1 mouse macrophages [Levitzki A, et al.(1980) Proc Natl Acad Sci USA 77: 2706-2710; Messick J B, et al. (1993)Infect Immun 61: 3803-3810; Schlegel R, et al. (1982) Proc Natl Acad SciUSA 79:2291-2295]. E. chaffeensis entry into THP-1 cells, leading toproductive infection, is dependent on the host-cell surface lipid raftsand glycosylphosphatidyl inositol (GPI)-anchored proteins [Lin M,Rikihisa Y (2003) Cell Microbiol 5: 809-820]. Furthermore, lipidraft-associated protein caveolin-1, but not clathrin localizes to the E.chaffeensis entry site [Lin M, Rikihisa Y (2003) Cell Microbiol 5:809-820]. After entry, E. chaffeensis replicates in the membrane-boundcompartment resembling an early endosome as it contains early endosomeantigen 1 (EEA1), Rab5, and transferrin receptor [Mott J, et al. (1999)Infect Immun 67: 1368-1378]. Several intracellular bacteria are known toenter host cells by using their specific surface protein collectivelycalled as ‘invasin’ or ‘internalin’ [Pizarro-Cerda J, et al. (2006) Cell124: 715-727]. However, detailed mechanisms of E. chaffeensis entry wereunknown; particularly regarding the involvement of any specificbacterial surface protein that can function as an invasin and itscognitive host-cell receptor [Rikihisa Y (2010) Nat Rev Microbiol 8:328-339].

The importance of E. canis as a veterinary pathogen in conjunction withthe recent identification of E. chaffeensis as the cause of an emergingtick-borne zoonosis has highlighted the need for improved diagnosticsand vaccines for both veterinary and human ehrlichioses, and thus theneed for identification of immunoreactive proteins.

SUMMARY

The comparative genome hybridization study of E. chaffeensis strainsidentified a protein (referred to herein as “entry triggering protein ofEhrlichia” or “EtpE”) with highly conserved N- and C-terminal segmentsflanking its strain-variable central region. As disclosed herein, EtpE,particularly its C-terminal conserved region (“EtpE-C”), is critical forEhrlichia sp. binding, entry, and infection of several different hostcell types. Immunization with rEtpE-C is also shown to protect miceagainst Ehrlichia sp. challenge. Further, antibodies to both the EtpE-Cand the N-terminal region (“EtpE-N”) can be found in subjects infectedwith Ehrlichia sp. Importantly, the EtpE-N is highly conserved acrossEhrlichia sp. Therefore, disclosed herein are vaccines, diagnostics, andcell delivery polypeptides and uses therefore that take advantage ofthese unique properties of EtpE.

In particular, a vaccine is disclosed that comprises one or morepolypeptides representing the EtpE from an Ehrlichia sp. and apharmaceutically acceptable adjuvant. For example, the one or morepolypeptide can comprise the amino acid sequence SEQ ID NO:1 (EtpE fromEhrlichia chaffeensis str. Arkansas; Accession No. YP_507823.1), SEQ IDNO:3 (EtpE from Ehrlichia canis str. Jake; Accession No. AAZ68869.1), orSEQ ID NO:5 (EtpE from Ehrlichia ruminantium str. Welgevonden; AccessionNo. YP_180660.1). Additional sequences for EtpE orthologues includeEhrlichia HF strain (Ixodes ovatus Ehrlichia) (Accession No.ABM69271.1), Ehrlichia chaffeensis Sapulpa strain (Accession No.ZP_00544864.1 and ZP_00544886.1), and Ehrlichia ruminantium, Gardelstrain (Accession No. YP_196760.1). EtpE homologues/orthologues fromother Ehrlichia sp. can also be identified and used to improve speciescross-reactivity.

In some embodiments, the vaccine contains an immunogenic fragment of anEtpE protein that is capable of eliciting an immune response against anEhrlichia sp. Preferably, the immunogenic fragment comprises at least aportion of the conserved region (“EtpE-C”). For example, the one or morepolypeptide can comprise the amino acid sequence SEQ ID NO:2 (residues1656-1963 of SEQ ID NO:1), SEQ ID NO:4 (residues 1408-1510 of SEQ IDNO:3), SEQ ID NO:6 (residues 1410-1710 of SEQ ID NO:5), or a animmunogenic fragment thereof capable of eliciting an immune responseagainst an Ehrlichia sp.

The vaccine can alternatively contain an immunogenic variant of an EtpEprotein, or fragment thereof, that is capable of eliciting an immuneresponse against an Ehrlichia sp. For example, the vaccine can compriseone or more polypeptides having an amino acid sequence with at least 70%identity to an amino acid sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or an immunogenic fragmentthereof capable of eliciting an immune response against an Ehrlichia sp.The vaccine can comprise one or more polypeptides having an amino acidsequence with at least 7% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, or a an immunogenic fragment thereof capable of eliciting animmune response against an Ehrlichia sp.

The vaccine comprises polypeptides representing the EtpE from at leastone Ehrlichia sp. However, to improve cross-reactivity, the vaccine cancontain polypeptides representing the EtpE from at least 2, 3, 4, 5, 6,or more Ehrlichia sp. These polypeptides can be in a single amino acidsequence, such as fusion protein. The polypeptides can also beconjugated together on a single carrier molecule. For example, thepolypeptides can be used in a multiple antigen peptide system (MAPS).

In some embodiments, the vaccine is capable of eliciting an immuneresponse against any combination of Ehrlichia chaffeensis, Ehrlichiacanis, and Ehrlichia ruminantium. The vaccine can also be capable ofeliciting an immune response against other species, such as Ehrlichiamuris, Ehrlichia ewingii, or a combination thereof. The EtpE homologuefrom these and other species and strains can be identified and used asimmunogens as described herein.

Also disclosed is a recombinant vector that contains a nucleic acidsequence encoding any of the one or more immunogenic polypeptidesdisclosed herein, operatively linked to a heterologous promoter.

Also disclosed is a vaccine containing any of the disclosed recombinantvectors in a pharmaceutically acceptable vehicle, diluent or excipient.The vaccine can further contain a pharmaceutically acceptable adjuvant.

Also disclosed is a method for vaccinating a subject against Ehrlichiasp, that comprises administering to the subject a composition comprisingany of the disclosed vaccines. The subject can be any mammal at risk forEhrlichia sp. infection. In particular, the subject can be a human,canine, feline, bovine, ovine, or caprine subject. The method provides aprotective immune response against at least one Ehrlichia sp. selectedfrom the group consisting of Ehrlichia chaffeensis, Ehrlichia canis, andEhrlichia ruminantium. However, in preferred embodiments, the vaccineelicits a protective immune response in the subject against at least 2,3, 4, 5, 6, or more Ehrlichia sp.

Also disclosed is a method for diagnosing Ehrlichiosis in a subject thatcomprising assaying a biological sample (e.g., blood, serum, or plasmasample) from the subject for the presence of an antibody thatspecifically binds an EtpE polypeptide. In particular, assaying forantibodies that specifically bind an EtpE-N provides pan-diagnosis ofEhrlichia sp. infection since the N-terminal domain is highly conservedacross species. Therefore, in some embodiments, the presence of theantibody is an indication that the subject has been infected with anEhrlichia sp. selected from the group consisting of Ehrlichiachaffeensis, Ehrlichia canis, Ehrlichia ruminantium, or any combinationthereof. Therefore, the method can involve assaying a biological samplefrom the subject for the presence of an antibody that specifically bindsSEQ ID NO:7 (amino acid residues 29-708 of SEQ ID NO:1), SEQ ID NO:8(amino acid residues 43-736 of SEQ ID NO:3), SEQ ID NO:9 (amino acidresidues 39-730 of SEQ ID NO:5), or a conservative variant thereofhaving at least 70% identity to SEQ ID NO:7, SEQ ID NO:8, or SEQ IDNO:9, wherein the presence of the antibody is an indication that thesubject has been infected with an Ehrlichia sp.

In some embodiments, the method provides a pan-diagnostic so that onetest can be used for multiple species of Ehrlichia. However, in someembodiments, the method also diagnoses the specific Ehrlichia sp. Inthese embodiments, the EtpE-C polypeptide can be used to detectantibodies that selectively bind the C-terminal region of an EtpE from aspecific species of Ehrlichia.

The disclosed methods can further comprise treating the subject forEhrlichiosis if the antibody is detected. For example, the method cancomprise treating the subject with an effective amount and duration ofdoxycycline if the antibody is detected. This should be done as early aspossible, since at later stage of infection antibiotic became lesseffective in clearing bacteria or clinical signs. Therefore, earlydetection by the disclosed methods can improve treatment efficacy.

Also disclosed is a method for monitoring the treatment of a subject forEhrlichiosis that comprises assaying a biological sample from thesubject for levels of an antibody that specifically binds a polypeptidecomprising SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, or a conservativevariant thereof having at least 70% identity to SEQ ID NO:7, SEQ IDNO:8, or SEQ ID NO:9. In these methods, an at least 2-, 3-, or 4-foldtiter reduction in antibody levels is an indication that the treatmentis effective. Of course, with chronic stage disease, this can takeseveral months. Therefore, the method can involve assaying a sample fromthe subject every 1, 2, 3, 4, 5, or 6 weeks to monitor the treatment.Once antibody levels are no longer detectable, the method can furtherinvolve ceasing treatment. Alternatively, if antibody levels do notdecrease as expected, then the method can further comprising alteringthe treatment, such as by increasing dosages or selecting an alternativeantibiotic.

Also disclosed is a method for delivering a therapeutic or diagnosticagent to a cell in a subject that involves conjugating the agent, or adelivery vehicle comprising the agent, to a EtpE-C polypeptide. Forexample, the method can comprise administering to the subject acomposition comprising the agent, wherein the agent, or a deliveryvehicle comprising the agent, is conjugated to a delivery polypeptidecomprising an amino acid sequence having at least 70% identity to SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, or a fragment thereof capable of bindingDNase X, or to a nucleic acid encoding the polypeptide operably linkedto a promoter. In these embodiments, the polypeptide can comprise atleast 100, 101, 102, 103, 104, 105, 110, 120, 130, 140, 150, 160, 170,180, 190, or 200 amino acids of SEQ ID NO:2, SEQ ID NO:4, or SEQ IDNO:6. In some embodiments, the polypeptide comprises at least residues1658 to 1761 of SEQ ID NO:1, or a conservative variant thereof. In someembodiments, the polypeptide comprises at least residues 1408 to 1510 ofSEQ ID NO:3, or a conservative variant thereof. In some embodiments, thepolypeptide comprises at least residues 1410 to 1510 of SEQ ID NO:5, ora conservative variant thereof. The disclosed method can be used todeliver the agent to any cell expressing DNase X. In addition toleukocyte, endothelial cells, and kidney cells, DNase X is highlyexpressed in heart, brain, and placenta. Cells from these tissues can betargeted for EtpE-C-DNase X-mediated gene or drug delivery.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A-1F show that EtpE-C is exposed at the bacterial surface, andanti-EtpE-C neutralizes E. chaffeensis infection in vitro. FIG. 1A is aWestern blot analysis of E. chaffeensis-infected (Ech) and uninfectedDH82 cells at 60 h pi using anti-EtpE-N (a-EtpE-N) and anti-EtpE-C(a-EtpE-C). FIG. 1B shows double immunofluorescence labeling of E.chaffeensis-infected human primary macrophages derived from peripheralblood monocytes at 56 h pi. Cells were fixed with PFA, permeabilizedwith saponin, and labeled with anti-EtpE-C and anti-E. chaffeensis majorouter membrane protein P28. The white dashed line denotes the macrophagecontour. The boxed region indicates the area enlarged in the smallerpanels to the right. Merge/DIC: Fluorescence images merged withDifferential interference contrast image (DIC). A single z-plane (0.4 mmthickness) by deconvolution microscopy was shown. Scale bar, 2 mm. FIG.1C shows E. chaffeensis incubated with DH82 cells for 30 min and doubleimmunofluorescence labeled using anti-EtpE-C and anti-E. chaffeensis P28without permeabilization. DAPI was used to label DNA. Scale bar, 1 mm.FIG. 1D is a bar graph showing numbers of E. chaffeensis bound to RF/6Acells at 30 min pi. Host cell-free E. chaffeensis was pretreated withanti-EtpE-C or preimmune mouse serum and incubated with RF/6A cells for30 min. Unbound E. chaffeensis was washed away, cells were fixed withPFA, and E. chaffeensis labeled with anti-P28 without permeabilization.E. chaffeensis in 100 cells were scored. FIG. 1E is a bar graph showingnumbers of E. chaffeensis internalized into RF/6A cells at 2 h pi. E.chaffeensis was pretreated with anti-rEtpE-C or preimmune mouse serumand incubated with RF/6A cells for 2 h. To distinguish intracellularfrom bound E. chaffeensis, unbound E. chaffeensis was washed away andcells were processed for two rounds of immunostaining with anti-P28;first without permeabilization to detect bound but not internalized E.chaffeensis (AF555-conjugated secondary antibody) and second round withsaponin permeabilization to detect total E. chaffeensis, i.e., boundplus internalized (AF488-conjugated secondary antibody). E. chaffeensisin 100 cells was scored. The black bar represents total E. chaffeensisand the white bar represents internalized E. chaffeensis (total minusbound) (see also FIG. 9). FIG. 1F is a bar graph showing qPCR for E.chaffeensis 16S rDNA in RF/6A cells infected with E. chaffeensis at 48 hpi. E. chaffeensis was pretreated with anti-EtpE-C or preimmune mouseserum and used to infect RF/6A cells; cells were harvested at 48 h pi.qPCR for E. chaffeensis 16S rDNA was normalized with G3PDH DNA. Datarepresent the mean and standard deviation of triplicate samples and arerepresentative of three independent experiments. *Significantlydifferent (P<0.05).

FIGS. 2A-2C show EtpE is expressed by E. chaffeensis in HME patients andinfected dogs, and immunization with rEtpE-C protects mice against E.chaffeensis challenge. FIG. 2A shows SDS-PAGE analysis and GelCode Bluestaining of rEtpE-N (lane 1) and rEtpE-C (lane 2) (5 mg/lane). rEtpE-Nwas partially cleaved after its expression in E. coli and thus isvisualized as multiple bands. FIG. 2B shows Western blot analysis ofrEtpE-N (lane 1) and rEtpE-C (lane 2) (5 μg/lane) with HME patient sera(ID: 72088, MRL1-22, MRL1-40) or control human serum (Control), or serafrom dogs experimentally infected with E. chaffeensis (ID: CTUALJ,3918815, 1425) or control dog serum. The relative band intensity forrEtpE-N/rEtpE-C (75 kDa and 34 kDa bands) assessed by densitometry wasshown beneath the panels. FIG. 2C shows dot-plot analysis of E.chaffeensis load of the blood samples from rEtpE-C-immunized andplacebo-immunized mice at 5 days after E. chaffeensis challenge. qPCR ofE. chaffeensis 16S rDNA normalized to mouse G3PDH DNA. *Significantlydifferent (P<0.05).

FIGS. 3A-3D show rEtpE-C-coated beads enter macrophages by a pathwaysimilar to one that mediates E. chaffeensis entry. FIG. 3A is an imageshowing latex beads coated with rEtpE-C by anti-EtpE-C labeling underfluorescence microscopy. Scale bar, 1 μm. FIG. 3B shows fluorescence andphase contrast merged images of rEtpE-C-coated beads incubated withmouse BMDMs. Cells were pretreated with DMSO (solvent control), MDC,genistein, or PI-PLC for 45 min followed by trypsin treatment to removebeads that were not internalized. Scale bar, 10 μm. FIGS. 3C and 3D arebar graphs showing numbers of internalized rEtpE-C-coated (C) andnon-coated (D) beads/cell incubated with mouse BMDMs pretreated withMDC, genistein, or PI-PLC, relative to DMSO treatment (solvent control)set as 100. Data represent the mean and standard deviation of triplicatesamples and are representative of three independent experiments.*Significantly different (P<0.05).

FIGS. 4A-4G show rEtpE-C-coated latex beads bind and enternon-phagocytic host cells. FIG. 4A shows rEtpE-C-coated beads (arrows),but not rEtpE-N, rECH0825, or rGroEL-coated beads, bind and enter HEK293cells at 1 h pi. Scale bar, 10 μm. FIG. 4B is a bar graph showingquantitation of similar experiment as (A) by scoring beads in 100 cells.Data represent the mean and standard deviation of triplicate samples andare representative of three independent experiments. *Significantlydifferent (P<0.05). FIG. 4C is a scanning electron micrograph ofrEtpE-C-coated beads on the surface of RF/6A cells at 2 h pi. Notefilopodia-like extensions embracing the beads (arrows). Scale bar, 1 μm.FIG. 4D is a transmission electron micrograph of rEtpE-C-coated beadsbeing engulfed (left panel) and internalized (right panel) into RF/6Acells at 8 h pi. Note filopodia-like extensions embracing the beads(arrow). Scale bars, 0.5 μm (left) and 1 μm (right). FIG. 4E containsfluorescence and DIC images of rEtpE-C-coated beads in RF/6A cells.RF/6A cells were pretreated with DMSO (solvent), MDC, verapamil, orgenistein for 30 min at 37° C., then incubated with rEtpE-C-coated beadsfor 8 h in the presence of compounds, washed and treated with trypsin toremove beads bound on the surface. A single z-plane (0.4 μm thickness)by deconvolution microscopy is shown here. Scale bar, 10 μm. FIG. 4F isa bar graph showing numbers of internalized rEtpE-C-coated beads/cell ofsimilar experiments as (E), relative to the number in DMSO treatment setas 100. Data represent the mean and standard deviation of triplicatesamples and are representative of three independent experiments.*Significantly different (P<0.05). FIG. 4G contains fluorescence and DICmerged images of RF/6A cells incubated with rEtpE-C-coated beadsimmunostained at 1 h pi with anti-EEA1 after permeabilization. Arrowsindicate beads surrounded with EEA1. The boxed region is enlarged to theright. A single z-plane (0.2 μm thickness) by deconvolution microscopyis shown here. Scale bar, 5 μm.

FIGS. 5A-5F show EtpE-C binds DNase X. FIG. 5A shows far-Westernblotting of renatured rEtpE-C and rECH0825 on a nitrocellulose membraneincubated with THP-1 cell lysate. Native DNase X was detected withanti-DNase X (α-DNase X), and recombinant proteins were detected withanti-histidine-tag (α-His tag). FIG. 5B shows Western blotting of THP-1cell lysate following affinity pull-down with rEtpE-C bound to Ni-silicamatrix. Bound proteins were eluted with imidazole and labeled witha-DNase X or α-His tag. FIG. 5C shows Western blot analysis of E.chaffeensis-infected THP-1 cell lysate immunoprecipitated withanti-EtpE-C (α-EtpE-C) or control IgG. THP-1 cells were incubated withE. chaffeensis for 30 min, followed by lysis, and immunoprecipitatedwith α-EtpE-Cor control mouse IgG-bound protein A agarose. Theprecipitates were subjected to Western blotting with α-DNase X. ** DNaseX, * mouse IgG heavy chain. FIG. 5D shows immunofluorescence labeling ofrEtpE-C-coated latex beads incubated with RF/6A cells for 1 h witha-DNase X without permeabilization. Note a cluster of beads colocalizeswith host cell-surface DNase X. Scale bar, 5 μm. FIG. 5E shows selectedtime-lapse images (0 to 6:38 min) of rEtpE-C-coated beads attached toRF/6A cells expressing DNase X-GFP at 4° C., and time 0 min was set uponraising the temperature to 37° C. The white dashed line denotes theRF/6A cell contour. A single z-plane (0.4 μm thickness) by deconvolutionmicroscopy was shown. Scale bar, 2 μm. FIG. 5F shows line intensityprofile analysis of rEtpE-C-beads and DNase X-GFP signal along thelength of the line (slanted white line in the image 5E).

FIGS. 6A-6D show internalization of rEtpE-C-coated beads is dependent onDNase X. FIG. 6A shows immunofluorescence labeling of rEtpE-C-coated ornoncoated beads incubated with human macrophages derived from peripheralblood monocytes. At 30 min pi, cells were labeled with a-DNase X withoutpermeabilization. rEtpE-C-coated beads cluster and colocalize with DNaseX on the cell surface, but non-coated beads do not. A single z-plane(0.4 μm thickness) by deconvolution microscopy was shown. Scale bar, 5μm (see also FIG. 13). FIG. 6B is a selected image showing theorthogonal view of macrophage incubated with rEtpE-C-coated (left panel)or non-coated (right panel) beads in (A). The orthogonal view wasobtained from the reconstituted 3-D view of serial z-stack images(combined z-section width of 7.2 μm). Scale bar, 5 μm. The fluorescenceintensity profiles of DNase X and beads signals were shown. FIG. 6Cshows fluorescence and phase contrast merged images of rEtpEC-coated andnon-coated beads incubated with BMDMs from DNase X^(−/−) and wild-typemice. Cells and beads were incubated for 45 min followed by trypsintreatment to remove non-internalized beads. Scale bar, 10 μm. FIG. 6Dshows numbers of internalized rEtpE-C-coated beads/cell of similarexperiment as (C), relative to the number of non-coated beads set as100. Data represent the mean and standard deviation of triplicatesamples and are representative of three independent experiments.*Significantly different (P<0.05) (see also FIG. 14).

FIGS. 7A-7J show that DNase X mediates E. chaffeensis binding, entry,and infection. FIG. 7A shows double immunofluorescence labeling witha-P28 and a-DNase X, without permeabilization, of E. chaffeensis boundon DH82 cells at 45 min pi at MOI of 10:1. The white dashed line denotesthe DH82 cell contour. The arrow indicates the area enlarged in thesmaller panels to the right. DNase X at the host-cell surface clustersto bound E. chaffeensis (arrows). Scale bar, 5 μm. FIG. 7B is a confocalimage of double immunofluorescence labeled E. chaffeensis on humanmacrophages derived from peripheral blood monocytes at 30 min pi at MOIof 10:1, with a-P28 and a-DNase X without permeabilization. DNase Xcolocalizes with the sites of E. chaffeensis binding (arrow, the regionenlarged in the smaller panels to the right). Scale bar, 5 μm. FIG. 7Cshows numbers of E. chaffeensis bound to DH82 cells pretreated witha-DNase X or mouse IgG at 30 min pi. Immunofluorescence labeling witha-P28 was performed without permeabilization and the numbers of E.chaffeensis on 100 cells were scored. Data represent the mean andstandard deviation of triplicate samples and are representative of threeindependent experiments. *Significantly different (P<0.05). FIG. 7Dshows numbers of E. chaffeensis internalized into DH82 cells pretreatedwith a-DNase X or mouse IgG at 2 h pi. Cells were processed for tworounds of immunostaining with a-P28 as described in FIG. 1E. The blackbar represents total E. chaffeensis, and the white bar representsinternalized E. chaffeensis (total minus bound). E. chaffeensis in 100cells were scored. Data represent the mean and standard deviation oftriplicate samples and are representative of three independentexperiments. *Significantly different (P<0.05). FIG. 7E shows E.chaffeensis load in DH82 cells pretreated with a-DNase X or mouse IgG at48 h pi. qPCR for E. chaffeensis 16S rDNA normalized with canine G3PDHDNA. Data represent the mean and standard deviation of triplicatesamples and are representative of three independent experiments.*Significantly different (P<0.05). FIG. 7F shows Western blot analysisof DNase X in HEK293 cells transfected with DNase X siRNA or scrambledcontrol siRNA. Actin was used as a protein loading control. FIG. 7Gshows E. chaffeensis load in HEK293 cells treated with DNase X siRNA orscrambled control siRNA at 48 h pi. qPCR for E. chaffeensis 16S rDNAnormalized with human G3PDH DNA. Data represent the mean and standarddeviation of triplicate samples and are representative of threeindependent experiments. *Significantly different (P<0.05). FIG. 7H is abar graph showing numbers of total cell-associated and internalized E.chaffeensis in DNase X^(−/−) or wild-type BMDMs at 4 h pi. Cells wereprocessed for two rounds of immunostaining with a-P28 as described inFIG. 1E. The total numbers of E. chaffeensis in 100 cells were scored.Data represent the mean and standard deviation of triplicate samples andare representative of three independent experiments. The black barrepresents total E. chaffeensis, and the white bar representsinternalized E. chaffeensis (total minus external). *Significantlydifferent (P<0.05) FIGS. 7I and 7J show E. chaffeensis load in BMDMsfrom DNase X^(−/−) mice and wild-type mice at 56 h pi (I) or in theblood at 5 days post-infection from DNase X^(−/−) mice and wild-typemice (J). qPCR for E. chaffeensis 16S rDNA was performed and normalizedwith mouse G3PDH DNA. Data represent the mean and standard deviation oftriplicate samples and are representative of three independentexperiments. *Significantly different (P<0.05).

FIG. 8 is a schematic representation of E. chaffeensis binding and entryinto mammalian cells. DNase X is enriched in the lipid raft domains ofthe cell membrane. Extracellular E. chaffeensis uses its surface proteinEtpE C-terminal region to make initial contacts with cell surface DNaseX that results in further lateral redistribution and local clustering ofDNaseX at the sites of bacterial binding. This binding elicits signalsthat are relayed down-stream and culminated in host cytoskeletalremodeling, filopodial induction and engulfment of the bound bacteriainto an early endosome into the host cell. This receptor-mediatedendocytosis can be specifically disrupted by genistein, verapamil orMDC. Latex beads coated with rEtpE-C also bind to cell surface DNase Xand follows a similar pattern of entry like that of E. chaffeensis.

FIG. 9 contains immunofluorescence image showing host cell-free E.chaffeensis that was either treated with pronase E or PBS control. Cellswere processed for double immunostaining with anti-EtpE-C and anti-CtrAwith or without saponin permeabilization as described to distinguishextracellular and internalized bacteria. When bacteria were treated withpronase E, the surface immunofluorescence staining of EtpE was abolishedcompletely, but not that of the internal control CtrA. Scale bar, 1 μm.

FIGS. 10A-10B show that anti-EtpE-C neutralizes E. chaffeensis bindingand entry into THP-1 cells, related to FIG. 1D-F. FIG. 10A is a bargraph showing numbers of E. chaffeensis (Ech) bound to THP-1 cells at 30min pi. E. chaffeensis was pretreated with anti-EtpE-C or preimmunemouse serum and incubated with THP-1 cells for 30 min. Unbound E.chaffeensis was washed away, cells were fixed with PFA and E.chaffeensis was labeled with anti-P28 without permeabilization. E.chaffeensis in 100 cells was scored. FIG. 10B is a bar graph showingnumbers of E. chaffeensis internalized into THP-1 cells at 2 h pi.Purified host cell-free E. chaffeensis was pretreated with anti-rEtpE-Cor preimmune mouse serum and incubated with THP-1 cells for 2 h. Todistinguish intracellular from bound E. chaffeensis, unbound E.chaffeensis was washed away, and cells were processed for two rounds ofimmunostaining with anti-P28: first without permeabilization to detectbound but not internalized E. chaffeensis (AF555-conjugated secondaryantibody), and another round with saponin permeabilization to detecttotal E. chaffeensis, i.e., bound plus internalized (AF488-conjugatedsecondary antibody). The black bar represents total E. chaffeensis, andthe white bar represents internalized E. chaffeensis (total minusbound). E. chaffeensis in 100 cells was scored. qPCR for E. chaffeensis16S rDNA was normalized with human G3PDH DNA. Data represent the meanand standard deviation of triplicate samples and are representative ofthree independent experiments. *Significantly different (P<0.05).

FIGS. 11A-11B show that anti-P28 does not inhibit binding or uptake ofE. chaffeensis by THP-1 cells, related to FIG. 1D-F. FIG. 11A is a bargraph showing relative radioactivity representing numbers of E.chaffeensis bound to THP-1 cells. Host cell-free radiolabeled E.chaffeensis preincubated with Fab fragment of rabbit anti-P28 IgG orpre-immune rabbit IgG were incubated with THP-1 cells for 2 h at 4° C.Unbound E. chaffeensis was washed away, and radioactivity of bound E.chaffeensis was measured. FIG. 11B is a bar graph showing relativeradioactivity representing numbers of E. chaffeensis internalized intoTHP-1 cells. Host cell-free radiolabeled E. chaffeensis preincubatedwith Fab fragment of rabbit anti-P28 IgG or pre-immune rabbit IgG wasincubated with THP-1 cells for 3 h at 37° C. Bound uninternalized E.chaffeensis was removed by pronase E treatment, radioactivity ofinternalized E. chaffeensis measured. Data represent the mean andstandard deviation of triplicate samples and are representative of twoindependent experiments.

FIGS. 12A-12B show that anti-EtpE-N is not effective in neutralizing E.chaffeensis infection in vitro and N-terminus of EtpE is lesssurface-accessible in live E. chaffeensis than its C-terminus, relatedto FIG. 1. FIG. 12A is a bar graph showing E. chaffeensis 16S rDNA inRF/6A cells infected with E. chaffeensis. E. chaffeensis was pretreatedwith anti-EtpE-N or preimmune rabbit serum and used to infect RF/6Acells; cells were harvested at 48 h pi. qPCR for E. chaffeensis 16S rDNAwas normalized with monkey G3PDH DNA. Data represent the mean andstandard deviation of triplicate samples and are representative of threeindependent experiments. *Significantly different (P<0.05). FIG. 12Bshows immunofluorescence labeling of live host cell-free E. chaffeensis.Unfixed E. chaffeensis was first incubated with anti-EtpE-C, EtpE-N, orP28 (ECHP28); then fixed and labeled with AF555-conjugated secondaryantibodies. Scale bar, 10 μm.

FIG. 13 shows that MDC blocks entry of E. chaffeensis into nonphagocyticRF/6A cells, related to FIG. 4E. Immunofluorescence labeling of E.chaffeensis incubated with RF/6A cells pretreated with MDC or DMSOcontrol. At 3 h pi, cells were treated with trypsin to removeun-internalized E. chaffeensis and then labeled with anti-P28. Scalebar, 10 μm. Bar graph shows quantitation by scoring E. chaffeensis (Ech)in 100 cells (right panel). Data represent the mean and standarddeviation of triplicate samples and are representative of threeindependent experiments. * Significantly different (P<0.05).

FIGS. 14A-14B show that rEtpE-C-coated beads recruit DNase X to theareas of binding, related to FIG. 6. rEtpE-C-coated or noncoated latexbeads were incubated with canine primary macrophages derived fromperipheral blood monocytes (A) or DH82 cells (B) at 37° C. for 30 min,and labeled with anti-DNase X without permeabilization. rEtpE-C-coatedbeads recruited surface exposed DNase X to their sites of binding andclustered, whereas non-coated beads did not colocalize with DNase X onthe cell surface. A single z-plane, of an optical section thickness of0.4-μm, at cell surface by deconvolution microscopy was shown. Scalebar, 5 μm.

FIGS. 15A-15B show that binding of rEtpE-C-coated beads is dependent onDNase X, related to FIGS. 6C and D. FIG. 15A contains fluorescence andDIC merged images of rEtpE-C-coated, rECH0825-coated and non-coatedbeads incubated with BMDMs from wild-type and DNase X^(−/−) mice. Beadswere incubated with cells for 30 min at 4° C. followed by rigorouswashing with PBS to remove unbound or loosely-adherent beads. Scale bar,5 μm. FIG. 15B is a bar graph showing numbers of internalizedrEtpE-C-coated beads/cell of similar experiment as (A), relative to thenumber of rECH0825-coated beads bound to wild-type BMDM set as 100. Datarepresent the mean and standard deviation of triplicate samples and arerepresentative of three independent experiments. *Significantlydifferent (P<0.05).

DETAILED DESCRIPTION

The comparative genome hybridization study of E. chaffeensis strainsrevealed that a hypothetical protein, ECH1038, consists of highlyconserved N- and C-terminal segments flanking its strain-variablecentral region. ECH1038 expression is up-regulated in the DC stage of E.chaffeensis. As disclosed herein, ECH1038 (here named as entrytriggering protein of Ehrlichia, EtpE), particularly its C-terminalconserved region (EtpE-C), is critical for E. chaffeensis binding,entry, and infection of several different host cell types. DNase X, ahost cell surface GPI-anchored protein, is the receptor of EtpE-Cmediating the entry of E. chaffeensis into several mammalian cell typespermissive to its replication. Moreover, immunization with rEtpE-C isalso shown herein to protect mice against Ehrlichia sp. challenge.Further, antibodies to both the EtpE-C and the N-terminal region(“EtpE-N”) can be found in subjects infected with Ehrlichia sp.Importantly, the EtpE-N is highly conserved across Ehrlichia sp.Therefore, disclosed herein are vaccines, diagnostics, and cell deliverypolypeptides and uses therefore that take advantage of these uniqueproperties of EtpEs.

Immunogenic polypeptides are disclosed that contain all or part of anEtpE protein from an Ehrlichia sp. This encompasses active fragments andvariants of the immunogenic polypeptide. Thus, the term “immunogenic orantigenic polypeptide” further contemplates deletions, additions andsubstitutions to the disclosed sequences, so long as the polypeptidefunctions to produce an immunological response as defined herein.

For example, the immunogenic polypeptide can comprise the amino acidsequence SEQ ID NO:1 (EtpE from Ehrlichia chaffeensis str. Arkansas;Accession No. YP_507823.1), SEQ ID NO:3 (EtpE from Ehrlichia canis str.Jake; Accession No. AAZ68869.1), or SEQ ID NO:5 (EtpE from Ehrlichiaruminantium str. Welgevonden; Accession No. YP_180660.1). EtpEhomologues from other Ehrlichia sp. can also be identified and used toimprove species cross-reactivity.

Importantly, genome sequencing has resulted in nomenclature changes andin some cases reclassification of both genus and species in the past. Itis therefore possible, that members of the Ehrlichia genus could bereclassified as a different species sometime in the future. Less likely,but still possible, is the future reclassification of a species in orout of the Ehrlichia genus. Therefore, while reference to species andgenus is meaningful, it is also understood that genus classification isless important than the presence of homologoues/orthologous EtpEproteins in the related organism, which can be identified and confirmedindependently from genus/species classification.

In some embodiments, the vaccine contains an immunogenic fragment of anEtpE protein that is capable of eliciting an immune response against anEhrlichia sp. Preferably, the immunogenic fragment comprises at least aportion of the conserved region (“EtpE-C”). For example, the one or morepolypeptide can comprise the amino acid sequence SEQ ID NO:2 (residues1656-1963 of SEQ ID NO:1), SEQ ID NO:4 (residues 1408-1510 of SEQ IDNO:3), SEQ ID NO:6 (residues 1410-1710 of SEQ ID NO:5), or a animmunogenic fragment thereof capable of eliciting an immune responseagainst an Ehrlichia sp.

The vaccine can alternatively contain an immunogenic variant of an EtpEprotein, or fragment thereof, that is capable of eliciting an immuneresponse against an Ehrlichia sp. For example, the vaccine can compriseone or more polypeptides having an amino acid sequence with at least70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identity to an amino acid sequence selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or animmunogenic fragment thereof capable of eliciting an immune responseagainst an Ehrlichia sp. The vaccine can comprise one or morepolypeptides having an amino acid sequence with at least 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or a an immunogenicfragment thereof capable of eliciting an immune response against anEhrlichia sp.

The vaccine comprises polypeptides representing the EtpE from at leastone Ehrlichia sp. However, to improve cross-reactivity, the vaccine cancontain polypeptides representing the EtpE from at least 2, 3, 4, 5, 6,or more Ehrlichia sp. These polypeptides can be in a single amino acidsequence, such as fusion protein. The polypeptides can also beconjugated together on a single carrier molecule. For example, thepolypeptides can be used in a multiple antigen peptide system (MAPS).

Fusion proteins, also known as chimeric proteins, are proteins createdthrough the joining of two or more genes which originally coded forseparate proteins. Translation of this fusion gene results in a singlepolypeptide with function properties derived from each of the originalproteins. Recombinant fusion proteins can be created artificially byrecombinant DNA technology for use in biological research ortherapeutics. Chimeric mutant proteins occur naturally when alarge-scale mutation, typically a chromosomal translocation, creates anovel coding sequence containing parts of the coding sequences from twodifferent genes.

A recombinant fusion protein is a protein created through geneticengineering of a fusion gene. This typically involves removing the stopcodon from a cDNA sequence coding for the first protein, then appendingthe cDNA sequence of the second protein in frame through ligation oroverlap extension PCR. That DNA sequence will then be expressed by acell as a single protein. The protein can be engineered to include thefull sequence of both original proteins, or only a portion of either.

If the two entities are proteins, often linker (or “spacer”) peptidesare also added which make it more likely that the proteins foldindependently and behave as expected. Especially in the case where thelinkers enable protein purification, linkers in protein or peptidefusions are sometimes engineered with cleavage sites for proteases orchemical agents which enable the liberation of the two separateproteins. This technique is often used for identification andpurification of proteins, by fusing a GST protein, FLAG peptide, or ahexa-his peptide (aka: a 6xhis-tag) which can be isolated using nickelor cobalt resins (affinity chromatography). Chimeric proteins can alsobe manufactured with toxins or anti-bodies attached to them in order tostudy disease development.

Alternatively, internal ribosome entry sites (IRES) elements can be usedto create multigene, or polycistronic, messages. IRES elements are ableto bypass the ribosome scanning model of 5′ methylated Cap dependenttranslation and begin translation at internal sites. IRES elements canbe linked to heterologous open reading frames. Multiple open readingframes can be transcribed together, each separated by an IRES, creatingpolycistronic messages. By virtue of the IRES element, each open readingframe is accessible to ribosomes for efficient translation. Multiplegenes can be efficiently expressed using a single promoter/enhancer totranscribe a single message.

In some embodiments, the vaccine is capable of eliciting an immuneresponse against at least Ehrlichia chaffeensis. In these embodiments atleast one of the one or more polypeptides comprise SEQ ID NO:1, SEQ IDNO:2, or an immunogenic variant or fragment thereof capable of elicitingan immune response against Ehrlichia chaffeensis.

In some embodiments, the vaccine is capable of eliciting an immuneresponse against at least Ehrlichia canis. In these embodiments at leastone of the one or more polypeptides comprise SEQ ID NO:3 or animmunogenic variant or fragment thereof capable of eliciting an immuneresponse against Ehrlichia canis.

In some embodiments, the vaccine is capable of eliciting an immuneresponse against at least Ehrlichia ruminantium. In these embodiments atleast one of the one or more polypeptides comprise SEQ ID NO:5 or animmunogenic variant or fragment thereof capable of eliciting an immuneresponse against Ehrlichia ruminantium.

In some embodiments, the vaccine is capable of eliciting an immuneresponse against any combination of Ehrlichia chaffeensis, Ehrlichiacanis, and Ehrlichia ruminantium. The vaccine can also be capable ofeliciting an immune response against other species, such as Ehrlichiamuris, Ehrlichia ewingii, or a combination thereof. The EtpE homologuefrom these and other species and strains can be identified and used asimmunogens as described herein.

Adjuvants suitable for use with the disclosed vaccine are known andinclude Quil A, aluminum salts, squalene, virosomes, and combinationsthereof. Likewise, immune stimulants suitable for use with the disclosedvaccine are known and include cytokines, growth factors, chemokines, andcombinations thereof.

Also disclosed is a recombinant vector that contains a nucleic acidsequence encoding any of the one or more immunogenic polypeptidesdisclosed herein, operatively linked to a heterologous promoter.Suitable vectors for propagating and/or expressing the nucleic acidsequence encoding any of the one or more immunogenic polypeptides areknown in the art. In some embodiments, the vector is a plasmid selectedfrom the group consisting of pcDNA3, pCI, VR1020, VR1012, and VR1055.These are mammalian expression plasmids which usually consist of astrong viral promoter to drive the in vivo transcription and translationof the gene (or complementary DNA) and include a strongpolyadenylation/transcriptional termination signal. For example, VR1055vector contains HCMV promotor, intron A (to improve mRNA stability) andmulticloning site, mRBG (minimal rabbit globin terminator) and kanamycinselection marker. A bacterial DNA sequence codon-optimized for mammalianexpression is inserted at the multicloning site.

In some embodiments, the vector is a recombinant viral vector. Forexample, the recombinant viral vector can be selected from the groupconsisting of poxvirus, adenovirus, adeno-associated virus, lentivirus,and herpesvirus. A live virus vector vaccine uses a weakened replicationdefective virus to transport genes encoding bacterial DNA sequence toelicit immune response to the bacterial protein. For example, a modifiedACAM2000 vaccinia virus (VACV) that is further reduced of its virulencepotential through deletion of thymidine kinase (TK; J2R) and IL-18binding protein (IL18BP; C12L). The inactivation of the TK gene greatlyreduces VACV virulence. In fact, a TK-null VACV, JX-594, was shown to besafe as an oncolytic virus for cancer patients in phase I clinicaltrial. Since viral envelop proteins elicit strong B- and T-cell immuneresponses, a fusion protein of viral envelop protein such as D8 and thebacterial protein is constructed and inserted into viral genome toproduce a recombinant virus.

Suitable promoters for driving expression of nucleic acids encoding thedisclosed polypeptides are also known and can be selected, for example,from the group consisting of human or murine cytomegalovirus majorimmediate early (IE) promoter (CMV-IE), the early/late promoter of theSV40 virus, and the LTR promoter of the Rous sarcoma virus. In someembodiments, a viral promoter can be used to drive a downstream geneencoding a fusion between a part of viral protein (for example, thetransmembrane domain of D8 of VACV and EtpE-C (codon optimized).

Also disclosed is a vaccine containing any of the disclosed recombinantvectors in a pharmaceutically acceptable vehicle, diluent or excipient.The vaccine can further contain a pharmaceutically acceptable adjuvant.Suitable adjuvants for use with DNA vaccines are known and include, forexample, an oligonucleotide comprising a CpG motif, or a vector encodingone or more growth factors, cytokines, chemokine, or combinationthereof. For example, the growth factor can be granulocyte macrophagecolony-stimulating factor (GM-CSF).

Also disclosed is a method for vaccinating a subject against Ehrlichiasp, that comprises administering to the subject a composition comprisingany of the disclosed vaccines. The subject can be any mammal at risk forEhrlichia sp. infection. In particular, the subject can be a human,canine, feline, bovine, ovine, or caprine subject. The method provides aprotective immune response against at least one Ehrlichia sp. selectedfrom the group consisting of Ehrlichia chaffeensis, Ehrlichia canis, andEhrlichia ruminantium. However, in preferred embodiments, the vaccineelicits a protective immune response in the subject against at least 2,3, 4, 5, 6, or more Ehrlichia sp.

Also disclosed is a method for diagnosing Ehrlichiosis in a subject thatcomprising assaying a biological sample (e.g., blood, serum, or plasmasample) from the subject for the presence of an antibody thatspecifically binds an EtpE polypeptide. In particular, assaying forantibodies that specifically bind an EtpE-N provides pan-diagnosis ofEhrlichia sp. infection since the N-terminal domain is highly conservedacross species. Therefore, in some embodiments, the presence of theantibody is an indication that the subject has been infected with anEhrlichia sp. selected from the group consisting of Ehrlichiachaffeensis, Ehrlichia canis, Ehrlichia ruminantium, or any combinationthereof. Therefore, the method can involve assaying a biological samplefrom the subject for the presence of an antibody that specifically bindsSEQ ID NO:7 (amino acid residues 29-708 of SEQ ID NO:1), SEQ ID NO:8(amino acid residues 73-736 of SEQ ID NO:3), SEQ ID NO:9 (amino acidresidues 39-730 of SEQ ID NO:5), or a conservative variant thereofhaving at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:7, SEQ ID NO:8,or SEQ ID NO:9, wherein the presence of the antibody is an indicationthat the subject has been infected with an Ehrlichia sp.

In some aspects, the method is an immunoassay. Many types and formats ofimmunoassays are known and all are suitable for detecting the disclosedbiomarkers. Examples of immunoassays are enzyme linked immunosorbentassays (ELISAs), radioimmunoassays (RIA), radioimmune precipitationassays (RIPA), immunobead capture assays, Western blotting, dotblotting, gel-shift assays, Flow cytometry, protein arrays, multiplexedbead arrays, magnetic capture, in vivo imaging, fluorescence resonanceenergy transfer (FRET), and fluorescence recovery/localization afterphotobleaching (FRAP/FLAP).

In some embodiments, the method provides a pan-diagnostic so that onetest can be used for multiple species of Ehrlichia. However, in someembodiments, the method also diagnoses the specific Ehrlichia sp. Inthese embodiments, the EtpE-C polypeptide can be used to detectantibodies that selectively bind the C-terminal region of an EtpE from aspecific species of Ehrlichia. This is useful since prognosis (choronicvs. acute disease) and zoonotic potential are different depending on thespecies of Ehrlichia. Although all of these species can infect humans,levels of zoonosis potential is in the order of E. chaffeensis>E.ewinigii>E. muris>E. canis>E. ruminantium. E. chaffeensis infection inhumans is acute, and can be fatal. E. canis infection is chronic anddebilitating febrile illness in dogs (infection for life if not treatedat early stage of infection). Clinical signs of E. canis infection inhumans is generally mild, but can be severe. E. canis can also infectcats, and clinical signs of cats are similar to those in infected dogs.E. ewingii infection in humans is acute, and some case is associatedwith infection of pet dogs (E. chaffeensis is not, since its majorreservoir is wild deer). E. ewingii infection of dogs is chronic. Wildrodents are the reservoir of E. muris (chronic infection), and clinicalsigns of human infection without underlining other diseases isrelatively mild, and has been transmitted between humans via bloodtransfusion of contaminated blood. E. ruminantium causes acute andchronic debilitating disease accompanied with neurological signs (heartwater, often fatal) of ruminants (cattle, goat, sheep) in Africa andCaribbean countries. Only rare cases of human infection with E.ruminantium (severe, acute) have been reported.

The disclosed methods can further comprise treating the subject forEhrlichiosis if the antibody is detected. For example, the method cancomprise treating the subject with an effective amount and duration ofdoxycycline if the antibody is detected. This should be done as early aspossible, since at later stage of infection antibiotic becomes lesseffective in clearing bacteria or clinical signs. Therefore, earlydetection by the disclosed methods can improve treatment efficacy.

Also disclosed is a method for monitoring the treatment of a subject forEhrlichiosis that comprises assaying a biological sample from thesubject for levels of an antibody that specifically binds a polypeptidecomprising SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, or a conservativevariant thereof having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:7,SEQ ID NO:8, or SEQ ID NO:9. In these methods, an at least 2-, 3-, or4-fold titer reduction in antibody levels is an indication that thetreatment is effective. Of course, with chronic stage disease, this cantake several months. Therefore, the method can involve assaying a samplefrom the subject every 1, 2, 3, 4, 5, or 6 weeks to monitor thetreatment. Once antibody levels are no longer detectable, the method canfurther involve ceasing treatment. Alternatively, if antibody levels donot decrease as expected, then the method can further comprisingaltering the treatment, such as by increasing dosages or selecting analternative antibiotic.

Also disclosed is a method for delivering a therapeutic or diagnosticagent to a cell in a subject that involves conjugating the agent, or adelivery vehicle comprising the agent, to a EtpE-C polypeptide. Forexample, the method can comprise administering to the subject acomposition comprising the agent, wherein the agent, or a deliveryvehicle comprising the agent, is conjugated to a delivery polypeptidecomprising an amino acid sequence having at least 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or a fragment thereofcapable of binding DNase X, or to a nucleic acid encoding thepolypeptide operably linked to a promoter.

In some embodiments, the polypeptide comprises at least 100, 101, 102,103, 104, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 aminoacids of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.

In some embodiments, the polypeptide comprises at least residues 1658 to1761 of SEQ ID NO:1, including residues 1658 to 1761, 1762, 1763, 1764,1765, 1766, 1767, 1768, 1769, 1770, 1771, 1772, 1773, 1774, 1775, 1776,1777, 1778, 1779, 1780, 1781, 1782, 1783, 1784, 1785, 1786, 1787, 1788,1789, 1790, 1791, 1792, 1793, 1794, 1795, 1796, 1797, 1798, 1799, 1800,1801, 1802, 1803, 1804, 1805, 1806, 1807, 1808, 1809, 1810, 1811, 1812,1813, 1814, 1815, 1816, 1817, 1818, 1819, 1820, 1821, 1822, 1823, 1824,1825, 1826, 1827, 1828, 1829, 1830, 1831, 1832, 1833, 1834, 1835, 1836,1837, 1838, 1839, 1840, 1841, 1842, 1842, 1844, 1845, 1846, 1847, 1848,1849, 1850, 1851, 1852, 1853, 1854, 1855, 1856, 1857, 1858, 1859, 1860,1861, 1862, 1863, 1864, 1865, 1866, 1867, 1868, 1869, 1870, 1871, 1872,1873, 1874, 1875, 1876, 1877, 1878, 1879, 1880, 1881, 1882, 1883, 1884,1885, 1886, 1887, 1888, 1889, 1890, 1891, 1892, 1893, 1894, 1895, 1896,1897, 1898, 1899, 1900, 1901, 1902, 1903, 1904, 1905, 1906, 1907, 1908,1910, 1911, 1912, 1913, 1914, 1915, 1916, 1917, 1918, 1919, 1920, 1921,1922, 1923, 1924, 1925, 1926, 1927, 1928, 1929, 1930, 1931, 1932, 1933,1934, 1935, 1936, 1937, 1938, 1939, 1940, 1941, 1942, 1943, 1944, 1945,1946, 1947, 1948, 1949, 1950, 1951, 1952, 1953, 1954, 1955, 1956, 1957,1958, 1959, 1960, 1961, 1962, or 1963 of SEQ ID NO:1, or a conservativevariant thereof.

In some embodiments, the polypeptide comprises at least residues 1408 to1510 of SEQ ID NO:3. In some embodiments, the polypeptide comprises atleast residues 1410 to 1510 of SEQ ID NO:5, including residues 1408 to1510,

1511, 1512, 1513, 1514, 1515, 1516, 1517, 1518, 1519, 1520, 1521, 1522,1523, 1524, 1525, 1526, 1527, 1528, 1529, 1530, 1531, 1532, 1533, 1534,1535, 1536, 1537, 1538, 1539, 1540, 1541, 1542, 1542, 1544, 1545, 1546,1547, 1548, 1549, 1550, 1551, 1552, 1553, 1554, 1555, 1556, 1557, 1558,1559, 1560, 1561, 1562, 1563, 1564, 1565, 1566, 1567, 1568, 1569, 1570,1571, 1572, 1573, 1574, 1575, 1576, 1577, 1578, 1579, 1580, 1581, 1582,1583, 1584, 1585, 1586, 1587, 1588, 1589, 1590, 1591, 1592, 1593, 1594,1595, 1596, 1597, 1598, 1599, 1600, 1601, 1602, 1603, 1604, 1605, 1606,1607, 1608, 1610, 1611, 1612, 1613, 1614, 1615, 1616, 1617, 1618, 1619,1620, 1621, 1622, 1623, 1624, 1625, 1626, 1627, 1628, 1629, 1630, 1631,1632, 1633, 1634, 1635, 1636, 1637, 1638, 1639, 1640, 1961, 1662, 1663,1644, 1645, 1646, 1647, 1648, 1649, 1650, 1651, 1652, 1653, 1654, 1655,1656, 1657, 1658, 1659, 1660, 1661, 1662, 1663, 1664, 1665, 1666, 1667,1668, 1669, 1670, 1671, 1672, 1673, 1674, 1675, 1676, 1677, 1678, 1679,1680, 1681, 1682, 1683, 1684, 1685, 1686, 1687, 1688, 1689, 1690, 1691,1692, 1693, 1694, 1695, 1696, 1697, 1698, 1699, 1700, 1701, 1702, 1703,1704, 1705, 1706, 1707, 1708, 1709, or 1710 of SEQ ID NO:5.

The therapeutic or diagnostic agent can be any pharmaceuticallyacceptable substance for which delivery to a cell in a subject isdesired. The agent can be a therapeutic drug (e.g., small molecule) orbiologic (e.g., antibody, peptide, growth factor). The diagnostic agentcan be a molecule detectable in the body of a subject by an imagingtechnique such as X-ray radiography, ultrasound, computed tomography(CT), single-photon emission computed tomography (SPECT), magneticresonance imaging (MRI), positron emission tomography (PET), OpticalFluorescent Imaging, Optical Visible light imaging, and nuclear medicineincluding Cerenkov Light Imaging. For example, the diagnostic agent cancomprise a radionuclide, paramagnetic metal ion, or a fluorophore.Fluorophores emit energy throughout the visible spectrum; however, thebest spectrum for in vivo imaging is in the near-infrared (NIR) region(650 nm-900 nm). Unlike the visible light spectrum (400-650 nm), in theNIR region, light scattering decreases and photo absorption byhemoglobin and water diminishes, leading to deeper tissue penetration oflight. Furthermore, tissue auto-fluorescence is low in the NIR spectra,which allows for a high signal to noise ratio. There is a range of smallmolecule organic fluorophores with excitation and emission spectra inthe NIR region. Some, such as indocyanine green (ICG) and cyaninederivatives Cy5.5 and Cy7, have been used in imaging for a relativelylong time. Modern fluorophores are developed by various biotechnologycompanies and include: Alexa dyes; IRDye dyes; VivoTag dyes andHylitePlus dyes. In general, the molecular weights of these fluorophoresare below 1 kDa. In some embodiments, the diagnostic agent comprises aradiocontrast agent. Examples of suitable radiocontrast agents includeiohexol, iodixanol and ioversol.

The disclosed delivery polypeptide can be conjugated to agents usingknown techniques, depending on the type of agent selected. For example,where the agent is a polypeptide, the composition can be a fusionprotein that contains both the agent and the delivery polypeptide.Likewise, the composition can comprises a DNA expression vector encodinga fusion protein comprising the agent and the delivery polypeptideoperably linked to a promoter. In some embodiments, the compositioncomprises a nanoparticle, microparticle, or microsphere encapsulatingthe agent. In these embodiments, the delivery polypeptide can bepositioned on the surface of the nanoparticle, microparticle, ormicrospheres to facilitate delivery. For example, the microsphere cancomprise lactide-co-glycoid or a polyanhydride. In some embodiments, thecomposition comprises a biodegradable polymer conjugated to the deliverypolypeptide.

The disclosed method can be used to deliver the agent to any cellexpressing DNase X. In addition to leukocyte, endothelial cells, andkidney cells, DNase X is highly expressed in heart, brain, and placenta.Cells from these tissues can be targeted for EtpE-C-DNase X-mediatedgene or drug delivery. A key premise for the success of cardiac genetherapy is the development of powerful gene transfer vehicles that canachieve highly efficient and persistent gene transfer specifically inthe heart. For example, the delivery mechanism can be used to delivervascular endothelial growth factor DNA to stimulate stem cells ofcardiac and skeletal muscles in vivo or to grow the cells in cellculture system to transplant matured muscle cells to regenerate thedamaged tissue. Leukemia cells can be treated through autologoustransplantation of hematopoietic stem cells gene-modified in vitro bydelivering a normal gene into hematopoietic stem cells.

Disclosed are pharmaceutical compositions containing therapeuticallyeffective amounts of one or more of the disclosed polypeptides, nucleicacids, or vaccines and a pharmaceutically acceptable carrier. The phrase“pharmaceutically acceptable” is employed herein to refer to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problems or complicationscommensurate with a reasonable benefit/risk ratio. Pharmaceuticalcarriers suitable for administration of the molecules provided hereininclude any such carriers known to those skilled in the art to besuitable for the particular mode of administration.

The polypeptides, nucleic acids, or vaccines can be formulated for avariety of routes of administration and/or applications. Suitable dosageforms for parenteral administration include solutions, suspensions, andemulsions. Typically, the polypeptides, nucleic acids, or vaccines aredissolved or suspended in a suitable solvent such as, for example,water, Ringer's solution, phosphate buffered saline (PBS), or isotonicsodium chloride. The formulation may also be a sterile solution,suspension, or emulsion in a nontoxic, parenterally acceptable diluentor solvent such as 1,3-butanediol. Formulations may further include oneor more additional excipients. Representative excipients includesolvents, diluents, pH modifying agents, preservatives, antioxidants,antinfective agents, suspending agents, wetting agents, viscositymodifiers, tonicity agents, stabilizing agents, and combinationsthereof. Suitable pharmaceutically acceptable excipients are preferablyselected from materials which are generally recognized as safe (GRAS),and may be administered to an individual without causing undesirablebiological side effects or unwanted interactions. In some cases,formulations can include one or more tonicity agents to adjust theisotonic range of the formulation. Suitable tonicity agents are wellknown in the art and include glycerin, mannitol, sorbitol, sodiumchloride, and other electrolytes. In some cases, the formulations can bebuffered with an effective amount of buffer necessary to maintain a pHsuitable for parenteral administration. Suitable buffers are well knownby those skilled in the art and some examples of useful buffers areacetate, borate, carbonate, citrate, and phosphate buffers. In someinstances, the formulation is distributed or packaged in a liquid form.Alternatively, formulations for ocular administration can be packed as asolid, obtained, for example by lyophilization of a suitable liquidformulation. The solid can be reconstituted with an appropriate carrieror diluent prior to administration.

The exact amount of the disclosed compositions administered to a subjectwill vary from subject to subject, depending on the nature of thediagnostic or therapeutic agent, the species, age, weight and generalcondition of the subject, the mode of administration and the like.Effective dosages and schedules for administering the compositions maybe determined empirically, and making such determinations is within theskill in the art. The dosage ranges for the administration of thecompositions are those large enough to produce the desired effect (e.g.,a therapeutic result or a suitable diagnostic result). The dosage shouldnot be so large as to cause adverse side effects, such as unwantedcross-reactions, anaphylactic reactions, and the like. The dosage can beadjusted by the individual physician in the event of anycounterindications.

The term “subject” refers to any individual who is the target ofadministration or treatment. The subject can be a vertebrate, forexample, a mammal. Thus, the subject can be a human or veterinarypatient. The term “patient” refers to a subject under the treatment of aclinician, e.g., physician.

The term “therapeutically effective” refers to the amount of thecomposition used is of sufficient quantity to ameliorate one or morecauses or symptoms of a disease or disorder. Such amelioration onlyrequires a reduction or alteration, not necessarily elimination.

The term “pharmaceutically acceptable” refers to those compounds,materials, compositions, and/or dosage forms which are, within the scopeof sound medical judgment, suitable for use in contact with the tissuesof human beings and animals without excessive toxicity, irritation,allergic response, or other problems or complications commensurate witha reasonable benefit/risk ratio.

The term “sample from a subject” refers to a tissue (e.g., tissuebiopsy), organ, cell (including a cell maintained in culture), celllysate (or lysate fraction), biomolecule derived from a cell or cellularmaterial (e.g. a polypeptide or nucleic acid), or body fluid from asubject. Non-limiting examples of body fluids include blood, urine,plasma, serum, tears, lymph, bile, cerebrospinal fluid, interstitialfluid, aqueous or vitreous humor, colostrum, sputum, amniotic fluid,saliva, anal and vaginal secretions, perspiration, semen, transudate,exudate, and synovial fluid.

The term “treatment” refers to the medical management of a patient withthe intent to cure, ameliorate, stabilize, or prevent a disease,pathological condition, or disorder. This term includes activetreatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder; preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder; and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder.

The terms “peptide,” “protein,” and “polypeptide” are usedinterchangeably to refer to a natural or synthetic molecule comprisingtwo or more amino acids linked by the carboxyl group of one amino acidto the alpha amino group of another.

The term “protein domain” refers to a portion of a protein, portions ofa protein, or an entire protein showing structural integrity; thisdetermination may be based on amino acid composition of a portion of aprotein, portions of a protein, or the entire protein.

The term “nucleic acid” refers to a natural or synthetic moleculecomprising a single nucleotide or two or more nucleotides linked by aphosphate group at the 3′ position of one nucleotide to the 5′ end ofanother nucleotide. The nucleic acid is not limited by length, and thusthe nucleic acid can include deoxyribonucleic acid (DNA) or ribonucleicacid (RNA).

A “fusion protein” refers to a polypeptide formed by the joining of twoor more polypeptides through a peptide bond formed between the aminoterminus of one polypeptide and the carboxyl terminus of anotherpolypeptide. The fusion protein can be formed by the chemical couplingof the constituent polypeptides or it can be expressed as a singlepolypeptide from nucleic acid sequence encoding the single contiguousfusion protein. A single chain fusion protein is a fusion protein havinga single contiguous polypeptide backbone. Fusion proteins can beprepared using conventional techniques in molecular biology to join thetwo genes in frame into a single nucleic acid, and then expressing thenucleic acid in an appropriate host cell under conditions in which thefusion protein is produced.

The term “specifically deliver” as used herein refers to thepreferential association of a molecule with a cell or tissue bearing aparticular target molecule or marker and not to cells or tissues lackingthat target molecule. It is, of course, recognized that a certain degreeof non-specific interaction may occur between a molecule and anon-target cell or tissue. Nevertheless, specific delivery, may bedistinguished as mediated through specific recognition of the targetmolecule. Typically specific delivery results in a much strongerassociation between the delivered molecule and cells bearing the targetmolecule than between the delivered molecule and cells lacking thetarget molecule.

The term “vector” refers to a replicon, such as a plasmid, phage, orcosmid, into which another DNA segment may be inserted so as to bringabout the replication of the inserted segment. The vectors can beexpression vectors.

The term “expression vector” refers to a vector that includes one ormore expression control sequences

The term “operably linked to” refers to the functional relationship of anucleic acid with another nucleic acid sequence. Promoters, enhancers,transcriptional and translational stop sites, and other signal sequencesare examples of nucleic acid sequences operably linked to othersequences. For example, operable linkage of DNA to a transcriptionalcontrol element refers to the physical and functional relationshipbetween the DNA and promoter such that the transcription of such DNA isinitiated from the promoter by an RNA polymerase that specificallyrecognizes, binds to and transcribes the DNA.

The terms “transformation” and “transfection” mean the introduction of anucleic acid, e.g., an expression vector, into a recipient cellincluding introduction of a nucleic acid to the chromosomal DNA of saidcell.

As used herein, the term “amino acid sequence” refers to a list ofabbreviations, letters, characters or words representing amino acidresidues. The amino acid abbreviations used herein are conventional oneletter codes for the amino acids and are expressed as follows: A,alanine; B, asparagine or aspartic acid; C, cysteine; D aspartic acid;E, glutamate, glutamic acid; F, phenylalanine; G, glycine; H histidine;I isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P,proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine;W, tryptophan; Y, tyrosine; Z, glutamine or glutamic acid.

As used herein, “peptidomimetic” means a mimetic of a peptide whichincludes some alteration of the normal peptide chemistry.Peptidomimetics typically enhance some property of the original peptide,such as increase stability, increased efficacy, enhanced delivery,increased half life, etc. Methods of making peptidomimetics based upon aknown polypeptide sequence is described, for example, in U.S. Pat. Nos.5,631,280; 5,612,895; and 5,579,250. Use of peptidomimetics can involvethe incorporation of a non-amino acid residue with non-amide linkages ata given position. One embodiment of the present invention is apeptidomimetic wherein the compound has a bond, a peptide backbone or anamino acid component replaced with a suitable mimic. Some non-limitingexamples of unnatural amino acids which may be suitable amino acidmimics include β-alanine, L-α-amino butyric acid, L-γ-amino butyricacid, L-α-amino isobutyric acid, L-ε-amino caproic acid, 7-aminoheptanoic acid, L-aspartic acid, L-glutamic acid,N-ε-Boc-N-α-CBZ-L-lysine, N-ε-Boc-N-α-Fmoc-L-lysine, L-methioninesulfone, L-norleucine, L-norvaline, N-α-Boc-N-δCBZ-L-ornithine,N-δ-Boc-N-α-CBZ-L-ornithine, Boc-p-nitro-L-phenylalanine,Boc-hydroxyproline, and Boc-L-thioproline.

The term “variant” refers to an amino acid or peptide sequence havingconservative amino acid substitutions, non-conservative amino acidsubstitutions (i.e. a degenerate variant), substitutions within thewobble position of each codon (i.e. DNA and RNA) encoding an amino acid,amino acids added to the C-terminus of a peptide, or a peptide having60%, 70%, 80%, 90%, or 95% homology to a reference sequence.

Variants also include allelic variants. The term “allelic variant”refers to a polynucleotide or a polypeptide containing polymorphismsthat lead to changes in the amino acid sequences of a protein and thatexist within a natural population (e.g., a virus species or variety).Allelic variants can be identified by sequencing the nucleic acidsequence of interest in a number of different species, which can bereadily carried out by using hybridization probes to identify the samegene genetic locus in those species. Any and all such nucleic acidvariations and resulting amino acid polymorphisms or variations that arethe result of natural allelic variation and that do not alter thefunctional activity of gene of interest, are intended to be within thescope of the disclosed polypeptides and nucleic acids.

The term “percent (%) sequence identity” or “homology” is defined as thepercentage of nucleotides or amino acids in a candidate sequence thatare identical with the nucleotides or amino acids in a reference nucleicacid sequence, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. Alignmentfor purposes of determining percent sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN,ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full-length of the sequences being compared can bedetermined by known methods.

The term “antibody” refers to natural or synthetic antibodies thatselectively bind a target antigen. The term includes polyclonal andmonoclonal antibodies. In addition to intact immunoglobulin molecules,also included in the term “antibodies” are fragments or polymers ofthose immunoglobulin molecules, and human or humanized versions ofimmunoglobulin molecules that selectively bind the target antigen.

The term “specifically binds”, as used herein, when referring to apolypeptide (including antibodies) or receptor, refers to a bindingreaction which is determinative of the presence of the protein orpolypeptide or receptor in a heterogeneous population of proteins andother biologics. Thus, under designated conditions (e.g. immunoassayconditions in the case of an antibody), a specified ligand or antibody“specifically binds” to its particular “target” (e.g. an antibodyspecifically binds to an endothelial antigen) when it does not bind in asignificant amount to other proteins present in the sample or to otherproteins to which the ligand or antibody may come in contact in anorganism. Generally, a first molecule that “specifically binds” a secondmolecule has an affinity constant (Ka) greater than about 10⁵ M⁻¹ (e.g.,10⁶ M⁻¹, 10⁷ M⁻¹, 10⁸ M⁻¹, 10⁹ M⁻¹, 10¹⁰ M⁻¹, 10¹¹ M⁻¹, and 10¹² M⁻¹ ormore) with that second molecule.

The term “immunogenic” as used herein refers to a composition that isable to provoke an immune response against it.

The term “immune response” as used herein refers to the reaction of theimmune system to the presence of an antigen by making antibodies to theantigen. In further specific embodiments, immunity to the antigen may bedeveloped on a cellular level, by the body as a whole, hypersensitivityto the antigen may be developed, and/or tolerance may be developed, suchas from subsequent challenge. In specific embodiments, an immuneresponse entails lymphocytes identifying an antigenic molecule asforeign and inducing the formation of antibodies and lymphocytes capableof reacting with it and rendering it less harmful.

The term “immunoreactive” as used herein refers to a composition beingreactive with antibodies from the sera of an individual. In specificembodiments, a composition is immunoreactive if an antibody recognizesit, such as by binding to it.

The term “ortholog” as used herein refers to a polynucleotide from onespecies that corresponds to a polynucleotide in another species; the twopolynucleotides are related through a common ancestral species (ahomologous polynucleotide). However, the polynucleotide from one specieshas evolved to become different from the polynucleotide of the otherspecies.

The term “vaccine” as used herein refers to a composition that providesimmunity to an individual upon challenge.

The term “subunit vaccine” as used herein refers to a vaccine wherein apolypeptide or fragment thereof is employed, as opposed to an entireorganism.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

EXAMPLES Example 1 ECH1038 (EtpE) is Highly Expressed by E. chaffeensisin Mammalian Cells

Materials and Methods

E. chaffeensis and Host Cell Culture.

E. chaffeensis Arkansas type strain was propagated in DH82 cells, andhost cell-free E. chaffeensis was obtained by controlled sonication asdescribed [Rikihisa Y, et al. (1994) J Clin Microbiol 32: 2107-2112;Barnewall R E, et al. (1994) Infect Immun 62: 4804-4810]. Humanperipheral blood monocytes were derived from buffy coats; HEK293, RF/6Aand THP-1 cells were cultured as previously described [Liu H, et al.(2012) Cell Microbiol 14: 1037-1050; Miura K, et al. (2011) Infect Immun79: 4947-4956; Barnewall R E, et al. (1994) Infect Immun 62: 4804-4810].BMDMs were established from wild-type and DNase X^(−/−) mice asdescribed [Miura K, et al. (2011) Infect Immun 79: 4947-4956].

Production of Recombinant Proteins rEtpE-N and rEtpE-C, and AntiseraAgainst them.

DNA fragments encoding EtpE-C and EtpE-N were amplified by PCR withPhusion high-fidelity DNA polymerase (NEB) using E. chaffeensischromosomal DNA as template. The fragments were cloned into pET33b(+)vector (Novagen); recombinant proteins were expressed in E. coli BL21(DE3) and purified by Ni-affinity chromatography. The antibody againstrEtpE-C was produced in ICR mice (Harlan), and the antibody againstrEtpE-N was produced in rabbits.

Statistical Analysis.

Statistical analysis was performed by unpaired two-tailed Student'st-test. P<0.05 was considered to be significant.

Results

ECH1038 (GenBank accession no. YP 507823, EtpE) consists of 1963 aminoacid residues (M_(r) 222,638, pI: 7.0) and is predicted to be an outermembrane protein with an N-terminal secretion signal by PSORT analysis[Miura K, Rikihisa Y (2007) Infect Immun 75: 3604-3613]. Although EtpEis variable in the central approximately 950 residues, the N-terminalapproximately 700 residues and C-terminal approximately 300 residues areconserved among multiple E. chaffeensis strains of distinct virulence,suggesting that these two regions are indispensable for E. chaffeensis.The amino acid sequence of EtpE orthologs in the three strains of E.chaffeensis, Arkansas, Wakulla, and Liberty are provided in SEQ ID NO:1(YP_507823), SEQ ID NO:10 (Accession No. DQ924562), and SEQ ID NO:11(Accession No. DQ924563), respectively. Amino acid sequence alignment ofEtpE orthologs among three genome-sequenced Ehrlichia species, E.chaffeensis Arkansas, E. canis Jake and E. ruminantium Welgevonden,revealed that while most of the N-terminal proximal region was conservedalso among Ehrlichia species, the C-terminal region was not.

To begin probing EtpE function, N-terminal (residues 29-708) andC-terminal (residues 1656-1963) of EtpE were cloned as C-terminalhistidine-tagged fusion proteins (rEtpE-N and rEtpE-C) and antisera wereprepared against rEtpE-N and rEtpE-C. Western blot analysis using theseantibodies showed that full-length EtpE was expressed by E. chaffeensisin DH82 cells (FIG. 1A). DH82 cells were initially used, since this cellline has been successfully used to culture isolate E. chaffeensis fromthe blood of HME patients [Dawson J E, et al. (1991) J Clin Microbiol29: 2741-2745; Paddock C D, et al. (1997) J Clin Microbiol 35:2496-2502].

To determine whether EtpE is expressed by E. chaffeensis in humanmonocytes, the pathogen's primary in vivo target cells, EtpE expressionwas determined in E. chaffeensis cultured in human primary macrophagesderived from peripheral blood monocytes by double immunofluorescencelabeling after paraformaldehyde (PFA) fixation and saponinpermeabilization. E. chaffeensis major outer membrane protein P28[Ohashi N, et al. (1998) Infect Immun 66: 132-139] was used as positivecontrol to label the bacterial membrane. The results showed that EtpEwas abundantly expressed by E. chaffeensis in human macrophages, andlocalized at bacterial membrane like P28 [Ohashi N, et al. (1998) InfectImmun 66: 132-139] (FIG. 1B).

Example 2 EtpE is Exposed on the E. chaffeensis Surface, and Anti-EtpE-CInhibits E. chaffeensis Binding, Entry, and Infection

Materials and Methods

Binding and Internalization Assay of E. chaffeensis and Immunostainingof Host Cell-Free E. chaffeensis.

Coverslip cultures of DH82, HEK293, RF/6A cells, or macrophagesdifferentiated from human peripheral blood monocytes or established frombone-marrow of DNase X^(−/−) or congenic wild-type mice and suspensionculture of THP-1 cells were incubated with E. chaffeensis freshlyisolated from infected cells at approximate multiplicity of infection(MOI) of 200, unless otherwise noted, for 30 to 45 min for bindingassays or 2 to 4 h for internalization assays at 37° C. in 5% CO²/95%air. Cells were washed with phosphate-buffered saline (PBS: 137 mM NaCl,2.7 mM KCl, 8.1 mM Na₂HPO₄, 1 mM KH₂PO₄, pH 7.4) to remove unboundbacteria and labeled with antibodies as described [Wang X, et al. (2006)Infect Immun 74: 1873-1882]. For binding assay cells were fixed with 3%PFA and labeled with mouse monoclonal anti-DNase X (Abcam), rabbitanti-E. chaffeensis P28 [Ohashi N, et al. (1998) Infect Immun 66:132-139], mouse anti-rEtpE-C, or dog anti-E. chaffeensis [Barnewall R E,et al. (1997) Infect Immun 65:1455-1461] as primary antibodies and AlexaFluor (AF) 488-conjugated goat anti-mouse IgG, AF555-conjugated goatanti-rabbit IgG (Invitrogen), or Texas Red-conjugated goat anti-dog IgG(Jackson ImmunoLab) as secondary antibodies. For internalization assays,two steps of labeling of fixed cells with anti-P28 were carried out asdescribed: the first labeling step was performed without saponinpermeabilization using AF488-conjugated goat anti-rabbit IgG, and thesecond labeling was performed with permeabilization usingAF555-conjugated goat anti-rabbit IgG [Niu H, et al. (2006) CellMicrobiol 8: 523-534]. Fluorescent images were acquired using a NikonEclipse E400 fluorescence microscope with a xenon-mercury light source(Nikon), Deltavision deconvolution microscope (Applied Precision) with0.2 or 0.4-μm step size along the z-axis of the cells, or an LSM 510laser-scanning confocal microscope (Carl Zeiss). For immunostaining oflive bacteria, host cell-free E. chaffeensis was incubated withanti-EtpE-C, anti-EtpE-N or E. chaffeensis P28 for 1 h at roomtemperature followed by fixing with 3% PFA and labeling withAF555-conjugated goat anti-mouse or anti-rabbit antibodies. To furtherdemonstrate the surface exposure of EtpE, host cell-free E. chaffeensiswas incubated with either pronase E (Sigma) at a concentration of 2mg/ml in PBS or vehicle control for 15 min at 37° C. [Yoshiie K, et al.(2000) Infect Immun 68: 1125-1133]; pronase E was inactivated by adding10% fetal bovine serum (FBS), followed by washing in PBS twice. Thebacteria were cytospun onto glass slides, fixed with 3% PFA, followed byquenching in PBS containing 0.1 M glycine, washed with PBS and labeledsequentially with anti-EtpE-C and anti-CtrA [Cheng Z, et al. (2011) MolMicrobiol 82: 1217-1234] with or without saponin permeabilizationfollowed by AF488 or AF555-conjugated goat anti-mouse or anti-rabbitantibodies.

[³⁵ S]Methionine-Labeled E. chaffeensis Binding and Uptake.

Approximately 10⁶ cells of E. chaffeensis-infected THP-1 cells/ml in 2ml of methionine cysteine-deficient RPMI 1640 medium (ICN Biomedicals)supplemented with 10% FBS and 2 mM L-Gln were incubated withcycloheximide (Sigma) at 10 μg/ml at 37° C. for 1 h. A metaboliclabeling reagent (Tran ³⁵S-Label; 11.7 mCi/ml [1,100 Ci/mmol]; 100 μl;ICN Biomedicals) was added and the mixture was incubated further at 37°C. for 24 h. The radiolabeled E. chaffeensis was released by sonicationand washed by centrifugation at 10,000×g for 10 min. To study the effectof rabbit anti-P28 on E. chaffeensis binding and entry, radiolabeled E.chaffeensis cells (40,000 cpm/200 μl) preincubated with Fab fragment ofanti-P28 IgG [0.5 mg/ml, prepared using Immobilized papain (Pierce) fromIgG affinity purified with AffiPack Immobilized Protein A column(Pierce)] or Fab fragment of normal rabbit IgG (0.5 mg/ml) were added to1×10⁶ THP-1 cells in 0.4 ml of RPMI 1640 medium containing 10% FBS and 2mM L-Gln and incubated at 4° C. for 2 h. The uptake of E. chaffeensiswas evaluated following removal of bound E. chaffeensis cells byincubation with pronase E at 2 mg/ml in PBS at 37° C. for 10 min afterincubation of E. chaffeensis with THP-1 cells at 37° C. for 3 h. THP-1cells were washed by centrifugation at 375×g for 5 min, the cells thenwere dissolved in 0.6 N NaOH and 0.5% SDS, and the radioactivity wasmeasured in a scintillation counter.

Results

P28 is bacterial surface exposed [Ohashi N, et al. (1998) Infect Immun66: 132-139] and is a β-barrel protein that functions as porin [KumagaiY, et al. (2008) J Bacteriol 190: 3597-3605]. To determine whether EtpEis exposed on the bacterial surface, double immunofluorescence labelingwith anti-EtpE-C and anti-P28 was performed after PFA fixation withoutsaponin permeabilization using E. chaffeensis bound to the surface ofDH82 cells. Unlike methanol or acetone fixation, PFA fixation does notallow antibody penetration across biological membranes unless withsubsequent permeabilization, thereby limiting antibody staining tomolecules exposed to the cell surface [Wang X, et al. (2006) InfectImmun 74: 1873-1882]. The result showed labeling of both EtpE and P28(FIG. 1C). Of note, labeled EtpE on E. chaffeensis had a beaded(rosary-like) pattern encircling individual bacterium, in contrast toP28 that had a uniform ring pattern (FIG. 1C). When host cell-freebacteria were treated with pronase E, the surface immunofluorescencestaining of EtpE was abolished, but not of CtrA which is an E.chaffeensis cytosolic response regulator of a two-component system[Cheng Z, et al. (2011) Mol Microbiol 82: 1217-1234] (FIG. 9). Thesedata indicate the surface exposure of EtpE. In contrast to the punctatelabeling pattern of EtpE in host cell-bound bacteria, homogeneouslabeling of EtpE was observed on host cell-free bacteria (FIG. 9).

Given the surface exposure of EtpE on E. chaffeensis, experiments wereconducted to determine whether the antibody against EtpE inhibitsbinding, entry, and infection of E. chaffeensis. Among the several hostcell types used in this study, primary monocytes, macrophages, andmyelocytic leukemia cell lines (DH82 and THP-1 cells) are referred to asphagocytes. Phagocytes are very efficient in bacteria and particleuptake as they have an array of dedicated phagocytic receptors,including pathogen pattern recognition receptors, mannose receptors,scavenger receptors, receptors for immunoglobulin (FcR) and complement(CR3) that utilize opsonins for ingestion, to name a few [Krieger M(1997) Curr Opin Lipidol 8: 275-280; Aderem A, et al. (1999) Annu RevImmunol 17: 593-623]. The other two cell lines used in this study, RF/6Aendothelial and HEK293 epithelial cells, are referred to asnon-phagocytes, since they lack these features. Non-phagocytes werefirst used to study the effect of in vitro antibody neutralization ofEtpE as they lack the response to opsonization and will not readilytake-up opsonized particles. E. chaffeensis was pre-incubated with mouseanti-EtpE-C or preimmune mouse sera, and then incubated with RF/6Acells. Binding and entry were determined by immunofluorescence labelingof E. chaffeensis with anti-P28 at 30 min and 2 hpost-incubation/infection (pi), respectively. Infection was determinedat 48 h pi by quantitative realtime PCR (qPCR). Anti-EtpE-C blocked E.chaffeensis binding, entry, and subsequently infection by approximately80% compared to preimmune serum (FIG. 1D-1F). Similar level ofinhibition of binding and entry was observed using mouse anti-EtpE-C inphagocytic cells such as human THP-1 cells (FIGS. 10A and 10B) andcanine DH82 cells. This suggests that human or canine FcR-mediated entryof E. chaffeensis opsonized with mouse anti-EtpE-C was negligible inthis experiment. Immunization of mice with recombinant P28, whichfunctions as a porin [Kumagai Y, et al. (2008) J Bacteriol 190:3597-3605], protects mice from E. chaffeensis challenge [Ohashi N, etal. (1998) Infect Immun 66: 132-139]. Additionally, in a mouse model ofHME, immunization of mice with Ehrlichia muris P28 conferred protectionfrom E. muris challenge [Crocquet-Valdes P A, et al. (2011) Clin VaccineImmunol 18: 2018-2025]. As another control, to rule out the possibilitythat inhibition of binding is a general property of antibodyneutralization of any E. chaffeensis cell surface proteins, experimentswere conducted to determine whether antibody against P28 blocks E.chaffeensis binding and entry. The result showed antibody (Fab fragment)against P28 did not block binding or entry of E. chaffeensis (FIGS. 11Aand 11B). Taken together, these results suggest that EtpE-C potentiallyserves as an invasin to trigger E. chaffeensis entry in both phagocytesand non-phagocytes.

EtpE is predicted to be anchored on the bacterial outer membrane at itsN-terminus, based on analysis using the PREDTMBB webserver [Miura K,Rikihisa Y (2007) Infect Immun 75: 3604-3613; Bagos P G, et al. (2004)Nucleic Acids Res 32: W400-404]. In contrast to anti-EtpE-C,anti-EtpE-N-pretreatment reduced E. chaffeensis infection by only 20%(FIG. 12A). Since both anti-EtpE-N and anti-EtpE-C reacted with nativeEtpE protein from E. chaffeensis equally well by Western blot analysis,accessibility of anti-EtpE-N to EtpE molecules was tested on live E.chaffeensis surface. For this purpose, the host cell-free E. chaffeensiswas freshly prepared and incubated with the antibodies withoutpre-fixation. The result showed that E. chaffeensis was not as readilylabeled with anti-EtpE-N as with anti-EtpE-C (FIG. 12B), suggesting thatthe antibody access to the N-terminal conserved region might be limitedin the native conformation of EtpE in live E. chaffeensis.

Example 3 EtpE is Expressed in HME Patients and Infected Dogs, andImmunization with rEtpE-C Suppresses E. chaffeensis Infection in Mice

Materials and Methods

Western Blot Analysis with Sera from HME Patients and E.chaffeensis-Infected Dogs.

Affinity-purified rEtpE-C and rEtpE-N (5 μg each) were subjected toSDS-PAGE, transferred to a nitrocellulose membrane, and incubated withsera from E. chaffeensis-infected dogs (CTUALJ, 3918815, 1425) [Huang H,et al. (2008) Infect Immun 76: 3405-3414], HME patients (ID: MRL1-22,MRL1-40, 72088) [Unver A, et al. (1999) J Clin Microbiol 37: 3888-3895],or control sera. After washing, the membranes were incubated withhorseradish peroxidase-conjugated goat anti-dog or anti-human IgG (KPL).Reacting bands were visualized with enhanced chemiluminescence (ECL),images were captured and densitometric analysis was performed using anLAS3000 image documentation system (FUJIFILM Medical Systems).

Mouse Infection Studies.

Two groups of C3H/HeJ mice (4-week-old females; 4 mice per group)(Jackson Laboratories) received either minced SDS-polyacrylamide gelcontaining 50 μg of rEtpE-C or minced gel alone, with Quil A (AccurateChemicals) as adjuvant for a total of three times at 14-day intervals.E. chaffeensis challenge was performed 10 days following the lastimmunization as described [Ohashi N, et al. (1998) Infect Immun 66:132-139]. DNase X^(−/−)[Rashedi I (2008) The Role of DNase X in SkeletalMuscle Addressed by Targeted Disruption of the Gene in a Mouse Model.Winnipeg: University of Manitoba. 122] and congenic wild-type C57BL/6mice (5- to 6-week-old females; 5 mice per group) were inoculatedintraperitoneally with E. chaffeensis-infected THP-1 cells (>90% cellsinfected; 6×10⁵ cells/mouse). DNA was extracted from blood samples usinga QIAamp blood kit (Qiagen), and subjected to qPCR using E. chaffeensis16S rDNA and mouse glyceraldehyde 3-phosphate dehydrogenase (G3PDH) geneprimers.

Results

Because EtpE is highly expressed by E. chaffeensis in mammalian cells invitro, experiments were conducted to determine whether EtpE is expressedin vivo by Western blot analysis of defined HME patient sera [Unver A,et al. (1999) J Clin Microbiol 37: 3888-3895]. Equal quantities ofrEtpE-N and rEtpE-C (GelCode Blue staining shown in FIG. 2A) were usedas antigens in the assay. Patient sera recognized both rEtpE-N andrEtpE-C, whereas the control serum from a healthy individual in an HMEnon-endemic region did not react with the recombinant proteins (FIG.2B). Similarly, sera from dogs experimentally infected with E.chaffeensis [Huang H, et al. (2008) Infect Immun 76: 3405-3414], thatwere previously shown to recognize E. chaffeensis OmpA [Cheng Z, et al.(2011) Mol Microbiol 82: 1217-1234] and other E. chaffeensislipoproteins [Huang H, et al. (2008) Infect Immun 76: 3405-3414],recognized both rEtpE-N and rEtpE-C, but the control dog serum did not(FIG. 2B). These data indicated that EtpE is expressed by E. chaffeensisin vivo during infection of its natural hosts, humans and dogs, and thatan antibody (humoral) response is mounted against this protein duringinfection and disease.

Antibodies contribute to immunity against E. chaffeensis inimmunocompetent mice [Yager E, et al. (2005) Infect Immun 73:8009-8016]. Given the facts that anti-EtpE-C neutralized E. chaffeensisbinding, consequently entry and infection in vitro, EtpE was expressedby E. chaffeensis in vivo and that a humoral immune response was mountedin infected mammals, experiments were conducted to examine whetherrEtpE-C immunization could confer protection in mice from E. chaffeensischallenge. C3H/HeJ strain of mice was used, since this strain wasreported to serve as a useful model for studying E. chaffeensisinfection [Telford S R (1996) Vet Microbiol 52: 103-112]. At 10 daysafter the last immunization, all mice were challenged intraperitoneallywith E. chaffeensis. The E. chaffeensis load in the blood fromrEtpE-C-immunized mice at 5 days post challenge was significantly lowerthan that of nonimmunized mice (FIG. 2C). These results indicate thatrEtpE-C is a protective immunogen relevant in E. chaffeensis infectionin vivo.

Example 4 Entry of rEtpE-C-Coated Beads into Macrophages is Blocked byCompounds that Block E. chaffeensis Invasion

Materials and Methods

Binding and Internalization of Latex Beads.

Sulfate-modified fluorescent red polystyrene beads (0.5 μm diameter;Sigma) at 3-4×10⁶ beads in 200 μl of 25 mM2-(N-morpholino)ethanesulfonic acid (MES) buffer, pH 8.0 were incubatedwith 1 mg of rEtpE-C or rEtpE-N proteins in 5-7 μl 7 M urea in 50 mMsodium phosphate buffer, pH 7.2 at 4° C. overnight with mixing at 20rpm. MES buffer (150 ml) was sequentially added to the mixture every 15min and incubated at room temperature, rotating at 20 rpm eventuallydiluting to around 200 times the original volume of urea buffer.rECH0825 and rGroEL were treated similarly, but without urea. The coatedbeads were collected by low speed centrifugation, washed twice in MESbuffer and re-suspended in complete DMEM or advanced MEM media, thengently sonicated to disperse the beads. Protein coating of the beadswere confirmed by dot blot assay and/or immunofluorescence labeling.Freshly prepared protein-coated or non-coated beads were added at amultiplicity of approximately 50 beads per cell for colocalizationstudies and 500 beads per cell for quantitation of binding andinternalization studies. The beads were incubated with HEK293 or RF/6Acells for 1 h at 37° C. Unbound beads were removed by washing and cellswere fixed for immunofluorescence labeling to detect the localization ofDNase X or EEA1 (anti-EEA1, BD). To study the effect of MDC, genistein,or verapamil on bead internalization, RF/6A cells were incubated withthese chemicals at a final concentration of 100 mM or 0.1% DMSO solventcontrol for 30 min, then with rEtpE-C-coated beads for 8 h at 37° C.BMDMs from wild-type mice were pre-incubated with MDC or genistein (100mM), PI-PLC (5 U/ml) or 0.1% DMSO for 30 min and then incubated withrEtpE-C-coated or non-coated beads for 45 min at 37° C. PI-PLC-treatedcells were washed prior to addition of rEtpE-C-coated beads. The cellswere washed and treated with 0.25% trypsin at 37° C. for 10 min toremove surface-bound beads. The detached cells were further washed bylow-speed centrifugation and later cytocentrifuged onto a glass slideand fixed with 3% PFA to observe internalized beads. To estimate thenumber of bound beads, a similar procedure for observing internalizedbeads was followed except that the beads were incubated with BMDM for 30min at 4° C. and following incubation the cells were washed to removeloosely bound beads and directly fixed with 3% PFA without trypsintreatment. For scanning electron microscopy, rEtpE-C-coated beads wereincubated with RF/6A cells for 2 h at 37° C. and processed as describedpreviously [Thomas S, et al. (2010) PLoS One 5: e15775]. Fortransmission electron microscopy, coated beads were incubated with RF/6Acells for 8 h at 37° C. and processed as described previously [RikihisaY, et al. (1979) J Exp Med 150: 703-708]. The 3D orthogonal view of thecell to show spatial distribution of DNase X with beads was obtained byusing the volume viewer function of SoftWoRx DeltaVision imageacquisition software from Applied Precision.

Live-Cell Imaging.

RF/6A cells were cultured in 35-mm glass bottom dishes (Wilco),transfected with DNase X-GFP, and incubated with rEtpE-C-coated beads at4° C. for 1 h to facilitate bead binding, but prevent internalization.Unbound beads were washed off, cells were replenished with mediumlacking phenol red, and the samples moved to a controlled environmentalchamber at 37° C. with under 5% CO²/95% air. Time-lapse images wereacquired at an interval of 10 s for 5 to 20 min through a 60×1.42 NA oilimmersion lens with an inverted Olympus IX-70 microscope, in 0.4-μmsteps in the z-axis using the attached Applied Precision motorized stage(DeltaVision deconvolution microscope). All stacks of images weredeconvoluted using SoftWoRx software and the time-lapse images of asingle focal plane of 0.4-μm focal depth at the cell surface wereexported as a video.

Results

Bacterial surface exposure of EtpE-C and effectiveness of EtpE-C as thetarget for both in vitro and in vivo neutralization suggest that EtpE-Cmay mediate E. chaffeensis invasion. To investigate this possibility,fluorescent latex beads of average size of 0.5 μm (similar to the sizeof infectious DCs of E. chaffeensis) were coated with rEtpE-C protein.The presence of rEtpE-C on beads was confirmed by dot-blot analysis andimmunofluorescence labeling with antiserum against EtpE-C (FIG. 3A).Beads were incubated with mouse bone marrow-derived macrophages (BMDMs)for 45 min followed by trypsin treatment to remove beads that were notinternalized. Mouse BMDMs were used here, also to serve as the wild-typecontrol for the later studies using BMDMs from mutant mice.rEtpE-C-coated beads entered BMDMs (FIGS. 3B and 3C). Treatment withMDC, genistein (broad-spectrum protein tyrosine kinase inhibitor), orphosphatidylinositol-specific phospholipase C (PI-PLC that removesGPI-anchored proteins from the cell surface) blocks E. chaffeensis entryand infection of THP-1 cells [Lin M, et al. (2002) Infect Immun 70:889-898; Lin M, Rikihisa Y (2003) Cell Microbiol 5: 809-820]. The entryof rEtpE-C-coated beads into BMDMs was almost completely blocked bythese treatments (FIGS. 3B and 3C), suggesting rEtpE-C-coated beadsenter BMDMs by the same signaling pathway as E. chaffeensis does. Thelatex bead is well-known to be taken up by macrophages and has been usedas a model to study phagocytosis [Werb Z, et al. (1972) J Biol Chem 247:2439-2446]. In striking contrast, entry of non-coated beads into BMDMswas not blocked by any of these treatments (FIG. 3D).

Example 5 rEtpE-C-Coated Beads Enter Non-Phagocytes

Results

Non-coated beads did not bind or enter RF/6A and HEK293 non-phagocyticcells (HEK293 cell data are shown in FIG. 4B). Remarkably,rEtpE-C-coated beads did readily bind and enter non-phagocytes (HEK293data are shown in FIGS. 4A and 4B). Beads coated with other recombinantE. chaffeensis proteins including rEtpE-N, rECH0825 (a type IV secretioneffector protein) [Liu H, et al. (2012) Cell Microbiol 14: 1037-1050] orrECH0365 (GroEL) did not bind HEK293 cells (FIGS. 4A and 4B), indicatingbinding and entry of beads into nonphagocytes was due to specificcoating with EtpE-C. Scanning and transmission electron microscopyrevealed that rEtpE-C-coated beads bound to RF/6A cells were associatedwith filopodia-like membrane projections (FIGS. 4C and 4D left panel)similar to those surrounding E. chaffeensis bound to DH82 cells [Zhang JZ, et al. (2007) Cell Microbiol 9: 610-618]. Transmission electronmicroscopy of RF/6A cells incubated with rEtpE-Ccoated beads verifiedthat the beads were indeed internalized into RF/6A cells (FIG. 4D rightpanel). MDC, genistein, and verapamil (a Ca²⁺ channel blocker) thatblock E. chaffeensis entry into THP-1 cells [Lin M, et al. (2002) InfectImmun 70: 889-898], also blocked E. chaffeensis entry into RF/6A cells(FIG. 13). Treatment with any of these compounds almost completelyblocked the entry of rEtpE-Ccoated beads into RF/6A cells (FIGS. 4E and4F). Once internalized, E. chaffeensis-containing vacuoles acquirecharacteristics of early endosomes [Mott J, et al. (1999) Infect Immun67: 1368-1378]. To determine whether rEtpE-C-coated beads were deliveredto early endosomes, immunofluorescence labeling was used to visualizethe spatial relationship of the early endosomal marker, EEA1 with therEtpE-C-coated beads, and observed by deconvolution microscopy.Individual as well as multiple beads were seen encased by EEA1-labeledmembranous compartment, suggesting that some beads were in endosomes(FIG. 4G). These results indicate that EtpE-C is an invasin, and even inthe absence of any other E. chaffeensis factors, EtpE-C alone issufficient to mediate the binding and entry of EtpE-C-coated beads intonon-phagocytic cells.

Example 6 EtpE-C Binds DNase X

Materials and Methods

Yeast Two-Hybrid Screening.

Yeast two-hybrid screening was performed using Matchmaker Two-HybridSystem (Clontech) according to manufacturer's instructions. The baitplasmid pGBKT7-EtpE-C was constructed by the fusion of EtpE-C with theGAL4 DNA-binding domain in pGBKT7 (Clontech) by PCR. EtpE-C codingsequence was amplified using the forward primer 5′-AATCCATGGA ATTGTTGTCATTAGTTGGTG GGCATCG-3′ (SEQ ID NO:12) and reverse primer 5′-TCGACGGATCCAATCCCCTT CCAGCATTAA TTTTATCAAA GG-3′ (SEQ ID NO:13), and the productwas ligated into pGBKT7. pGBKT7-EtpE-C was transformed intoSaccharomyces cerevisiae strain AH109 and selected by the ability togrow on SD agar plates lacking tryptophan. The expression of baitprotein EtpE-C in yeast was examined by Western blotting. The human bonemarrow MATCHMAKER cDNA library (Clontech) that was fused withGAL4-activating domain in pGADT7 was transformed in S. cerevisiae strainY187 (Clontech). Library clones expressing interacting prey proteinswere screened with yeast mating. Positive clones were selected by theirability to grow on SD quadruple drop-out (SD/QDO) plates lackingadenine, histidine, leucine, and tryptophan, and verified on SD/QDOplates containing X-gal. Positive clones were then isolated, and theprey plasmids were purified and sequenced after they were transformedinto E. coli TOP10F9 competent cells (Invitrogen). The interaction wasconfirmed by re-shuttling the purified prey plasmid into S. cerevisiaeAH109 transformed with bait plasmid and by nutritional selection inSD/QDO plates.

Far-Western Blotting, Protein Affinity Pull-Down andCoimmunoprecipitation.

Far-Western blotting was performed using 5 μg of rEtpE-C and rECH0825that were separated by SDS-PAGE, transferred to a nitrocellulosemembrane and renatured with serial guanidinium-HCl treatment followed byincubation with THP-1 cell lysate in NP-40 lysis buffer (150 mM NaCl, 50mM Tris-HCl pH 7.4, 1% w/v NP-40, supplemented with 1% proteaseinhibitor cocktail set III [Calbiochem]) as described [Bao W, et al.(2009) J Bacteriol 191: 278-286]. After stringent washing, the membranewas incubated with anti-DNase X and peroxidase-conjugated goatanti-mouse antibodies (KPL). The membrane was stripped with RestoreWestern Blot Stripping Buffer (Thermo scientific) and re-probed withperoxidase-conjugated anti-histidine antibody (Sigma). For proteinpull-down, His-tagged rEtpE-C was bound to and renatured on theNi-affinity matrix (Promega). THP-1 cell lysate in NP-40 lysis bufferwas applied to the matrix and incubated for 8 h at 4° C. After washingoff the unbound or non-specifically bound proteins from the matrix,rEtpE-C and bound protein complex were eluted with 250 mM imidazole. Theeluate and the post-elution Ni-matrix were resuspended in 26 SDS-samplebuffer and subjected to Western blotting with anti-DNase X antibody. Forco-immunoprecipitation assay, THP-1 cells were incubated with E.chaffeensis for 30 min and lysed in NP-40 lysis buffer. The lysate wasimmunoprecipitated with anti-EtpEC (2 μg)-bound protein A agarose orcontrol mouse IgG (2 μg)-bound agarose beads. The precipitate wasre-suspended in 2×SDS-sample buffer and subjected to Western blottingwith anti-DNase X antibody.

Results

Since EtpE-C could mediate binding and entry of EtpE-C-coated beads,Experiments were conducted to search for the potential host-cellreceptor for EtpE-C. EtpE-C was cloned into the yeast two-hybrid baitvector and a human bone marrow cDNA prey library was screened toidentify interacting proteins. Of the 5 clones detected and sequenced,all of them encoded a protein, deoxyribonuclease 1-like 1 (DNase 1L1,DN1L1, or DNase X on chromosome Xq28, GenBank accession no: X90392, 302residues). One of the clones contained an additional plasmid encodingS-adenosyl methionine-dependent methyltransferase but the codingsequence was out-of-frame; this prey construct alone did not supportyeast growth when co-transformed with bait vector to test theirinteraction. All sequence hits corresponded to the C-terminal fragmentof DNase X (residues 105-302). DNase X, one of the human DNase I-familyendonucleases, is expressed on the cell surface as a GPI-anchoredprotein and also localized at early endocytic vesicles, endoplasmicreticulum, and Golgi [Shiokawa D, et al. (2001) Biochemistry 40:143-152; Shiokawa D, et al. (2007) J Biol Chem 282:17132-17140].

To confirm EtpE-C binding to the native human DNase X, far-Western blotanalysis was performed. DNase X from the THP-1 cell lysate bound tore-natured rEtpE-C on a nitrocellulose membrane, whereas DNase X did notbind the control rECH0825 (FIG. 5A). Next, a protein pull-down assay wasused wherein THP-1 cell lysate was applied to rEtpE-C bound to andrenatured on a Ni-affinity matrix. Western blotting showed that nativeDNase X from the lysate bound to rEtpE-C, but not to the controlrECH0825 (FIG. 5B). In addition, co-immunoprecipitation showed thatanti-EtpE-C, but not the control mouse IgG pulled down native DNase Xfrom the lysate of THP-1 cells incubated with E. chaffeensis for 30 min(FIG. 5C). Taken together, these results indicate that EtpE-C can bindto DNase X.

RF/6A or HEK293 cells incubated with rEtpE-C-coated beads were fixed,and without membrane permeabilization, immunofluorescence labeled cellsurface-exposed DNase X was conducted. DNase X localized to the areas onthe surface of cells where rEtpE-C-coated beads were present (RF/6A cellimage shown in FIG. 5D). Timelapse live-cell image analysis ofrEtpE-C-coated beads bound to RF/6A cells ectopically expressing DNaseX-GFP at 4° C. showed that, upon warming up to 37° C., the initiallyseparated DNase X-GFP signal and beads became closer and overlappedwithin 5 min (FIG. 5E). The fluorescence intensity profile analysis ofred (rEtpE-C-coated beads) and green (DNase X-GFP) signal along thelength of the line also revealed that the signals separated at initialtime points converged in a few min after warming-up (FIG. 5F).

Example 7 Binding and Internalization of rEtpE-C-Coated Beads isDependent on DNase X

Results

Since DNase X localized to EtpE-C-coated beads in nonphagocytes, thisphenomenon was next examined in phagocytes. Human primary macrophagesderived from peripheral blood monocytes were incubated withrEtpE-C-coated or non-coated beads, cell surface exposed DNase X wasimmunofluorescence-labeled without permeabilization and the distributionof beads and DNase X was examined by deconvolution microscopy. SurfaceDNase X was seen clustered with rEtpE-C-coated beads; whereas bothsurface DNase X and beads were randomly dispersed with non-coated beads(image in a single z-plane shown in FIG. 6A). Orthogonal views of thecell from the reconstructed 3D view of serial z-stack imagesunequivocally demonstrated colocalization of DNase X with rEtpE-C-coatedbeads (FIG. 6B left panel), whereas DNase X did not colocalize withnon-coated beads (FIG. 6B right panel). The intensity profile analysisof green (DNase X) and red (beads) signals of a single optical sectionshowed that DNase X coincided with rEtpE-C-coated beads, but not withnoncoated beads (FIG. 6B right panels). Similar results were observedwith canine primary macrophages derived from peripheral blood monocytesand DH82 cells (FIG. 14). These results indicate DNase X localizes torEtpE-C-coated beads in primary human and canine macrophages, thepathogen's in vivo target cells.

rEtpE-C-coated beads entered wild-type mouse BMDMs as shown in FIGS. 3Band 3C. Therefore, experiments were conducted to determine whetherrEtpE-C-coated beads can enter BMDMs from congenic DNase X^(−/−) mice.Beads were incubated with BMDMs from DNase X^(−/−) mice for 45 minfollowed by trypsin treatment to remove beads that were notinternalized. Results showed rEtpE-C-coated beads did not enter DNaseX^(−/−) BMDMs (FIGS. 6C and 6D). In striking contrast, non-coated beadsfreely entered DNase X^(−/−) BMDMs (FIGS. 6C and 6D). This lack of entryof rEtpE-C-coated beads into DNase X^(−/−) BMDM, but not into thewild-type BMDM, was a direct consequence of its failure to bind DNaseX^(−/−) BMDM (FIG. 15). This phenomenon was specific to rEtpE-C-coatedbeads, because neither the non-coated beads nor the rECH0825-coatedbeads showed any defect in binding DNase X^(−/−) BMDMs (FIG. 15). Takentogether, these results indicate that rEtpE-C coating dictates the latexbead binding and entry via DNase X-dependent pathway.

Example 8 DNase X Mediates E. chaffeensis Binding, Entry, and Infection

Materials and Methods

In Vitro Neutralization and RNA Interference.

E. chaffeensis preincubated with 25 μg/ml of mouse anti-rEtpEC, rabbitanti-rEtpE-N, or preimmune mouse or rabbit sera for 1 h at 4° C. wereused to infect THP-1, RF/6A, or DH82 cells. Alternatively, E.chaffeensis was added to DH82 cells preincubated with 10 μg/ml ofmonoclonal anti-DNase X or control mouse monoclonal antibody for 30 minat 25° C. in serum-free DMEM. Binding, internalization, and infectionwere determined at 30 min, 2 h and 48 h pi, respectively. HEK293 cellsin 24-well plates were transfected with 50 nM DNase X siRNA (Santa CruzBiotechnology) or scrambled control siRNA using Lipofectamine 2000(Invitrogen). A second transfection with 50 nM of DNase X and scrambledsiRNAs was performed 30 h after the first transfection. An aliquot ofcells were harvested at 24 h after the second transfection to determinethe protein amount of DNase X by Western blotting and densitometryanalysis with anti-DNase X and rabbit anti-actin (Sigma). The otheraliquot of cells were incubated with E. chaffeensis and incubated for anadditional 48 h to evaluate infection. Infection was determined by qPCRof E. chaffeensis 16S rRNA gene relative to host cell G3PDH gene [ChengZ, et al. (2011) Mol Microbiol 82: 1217-1234].

Results

Since DNase X was localized to EtpE-C-coated bead entry foci,experiments were conducted to determine whether DNase X localizes to theE. chaffeensis entry foci as well. Double immunofluorescence labeling ofnon-permeabilized DH82 cells and primary human macrophages derived fromhuman peripheral blood monocytes showed surface DNase X colocalizationwith the bound E. chaffeensis (FIGS. 7A and 7B). DNase X also localizedto the areas of E. chaffeensis binding on THP-1 cells. When DH82 cellswere pre-incubated with monoclonal anti-DNase X IgG to block thesurface-exposed DNase X, E. chaffeensis binding, entry, and overallinfection were significantly reduced compared with the control mouseIgG-treated DH82 cells (FIGS. 7C-7E). Next, a small interfering RNA(siRNA) against DNase X was used to reduce the expression of endogenousDNase X in HEK293 cells (FIG. 7F). Suppression of DNase X expressionsignificantly reduced E. chaffeensis infection in HEK293 cells (FIG.7G). Moreover, E. chaffeensis binding and entry were reduced by 60% inDNase X^(−/−) BMDMs compared to the wild-type BMDMs (FIG. 7H). E.chaffeensis load at 56 h pi was significantly lower in DNase X^(−/−)BMDMs compared to the wild-type BMDMs (FIG. 7I). These resultsdemonstrated that effective E. chaffeensis binding, entry, and infectiondepended on DNase X. Importantly, E. chaffeensis load in peripheralblood at 5 days pi in DNase X^(−/−) mice was significantly lower than inwild-type mice (FIG. 7J), indicating that effective in vivo infection ofE. chaffeensis also requires involvement of DNase X.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A method for delivering a therapeutic ordiagnostic agent to a cell in a subject, comprising administering to thesubject a composition comprising the agent, wherein the agent, or adelivery vehicle comprising the agent, is conjugated to a deliverypolypeptide comprising an amino acid sequence having at least 70%identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or to a nucleic acidencoding the polypeptide operably linked to a promoter.
 2. The method ofclaim 1, wherein the composition comprises a fusion protein comprisingthe agent and the delivery polypeptide.
 3. The method of claim 1,wherein the composition comprises a DNA expression vector encoding afusion protein comprising the agent and the delivery polypeptideoperably linked to a promoter.
 4. The method of claim 1, wherein thecomposition comprises a nanoparticle, microparticle, or microsphereencapsulating the agent, wherein the delivery polypeptide is positionedon the surface of the nanoparticle, microparticle, or microspheres. 5.The method of claim 4, wherein the microsphere comprises(lactide-co-glycoid or polyanhydrides.
 6. The method of claim 1, whereinthe composition comprises a biodegradable polymers conjugated to thedelivery polypeptide.
 7. The method of claim 1, wherein the cellcomprises a monocyte.
 8. The method of claim 1, wherein the cellcomprises a heart or skeletal muscle cell.